Use of microfluidic systems in the electrochemical detection of target analytes

ABSTRACT

The invention relates generally to methods and apparatus for conducting analyses, particularly microfluidic devices for the detection of target analytes.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 09/295,691, filedApr. 21, 1999, the disclosure of which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The invention relates generally to methods and apparatus for conductinganalyses, particularly microfluidic devices for the detection of targetanalytes.

BACKGROUND OF THE INVENTION

There are a number of assays and sensors for the detection of thepresence and/or concentration of specific substances in fluids andgases. Many of these rely on specific ligand/antiligand reactions as themechanism of detection. That is, pairs of substances (i.e. the bindingpairs or ligand/antiligands) are known to bind to each other, whilebinding little or not at all to other substances. This has been thefocus of a number of techniques that utilize these binding pairs for thedetection of the complexes. These generally are done by labeling onecomponent of the complex in some way, so as to make the entire complexdetectable, using, for example, radioisotopes, fluorescent and otheroptically active molecules, enzymes, etc.

There is a significant trend to reduce the size of these sensors, bothfor sensitivity and to reduce reagent costs. Thus, a number ofmicrofluidic devices have been developed, generally comprising a solidsupport with microchannels, utilizing a number of different wells,pumps, reaction chambers, and the like. See for example EP 0637996 131;EP 0637998 B1; WO96/39260; WO97/16835; WO98/13683; WO97/16561;WO97/43629; WO96/39252; WO96/15576; WO96/15450; WO97i37755; andWO97/27324; and U.S. Pat. Nos. 5,304,487; 5,071,531; 5,061,336;5,747,169; 5,296,375; 5,110,745; 5,587,128; 5,498,392; 5,643,738;5,750,015; 5,726,026; 5,35,358; 5,126,022; 5,770,029; 5,631,337;5,569,364; 5,135,627; 5,632,876; 5,593,838; 5,585,069; 5,637,469;5,486,335; 5,755,942; 5,681,484; and 5,603,351. However, there is a needfor a microfluidic biosensor that can utilize electronic detection ofthe analytes.

However, there is a need for a microfluidic biosensor that can utilizeelectronic detection of the analytes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D depict some preferred embodiments of the invention. FIG. 1Adepicts a solid support 5 that has a sample inlet port 10, a firstmicrochannel 15, a storage module 25 (for example, for assay reagents)with a second microchannel 20. The second microchannel (20B) may be influid contact directly with the detection module 30 comprising adetection electrode 35, or (20A) in contact with the first microchannel15. FIG. 1B depicts a sample handling well 40 and a second storage well25A with a microchannel 20 to the sample handling well 40. For example,the sample handling well 40 could be a cell lysis chamber and thestorage well 25A could contain lysis reagents. FIG. 1C depicts a samplehandling well 40 that is a cell capture or enrichment chamber, with anadditional reagent storage well 25B for elution buffer. FIG. 1D depictsthe addition of a reaction module 45, with a storage module 25C forexample for storage of amplification reagents. Optional waste module 26is connected to the reaction module 45 via a microchannel 27. All ofthese embodiments may additionally comprise valves, waste wells, andpumps, including additional electrodes.

FIG. 2 depicts a cross-sectional view of a detection electrode accordingto one embodiment of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides microfluidic cassettes or devices that can beused to effect a number of manipulations on a sample to ultimatelyresult in target analytes detection or quantification. Thesemanipulations can include cell handling (cell concentration, cell lysis,cell removal, cell separation, etc.), separation of the desired targetanalyte from other sample components, chemical or enzymatic reactions onthe target analyte, detection of the target analyte, etc. The devices ofthe invention can include one or more wells for sample manipulation,waste or reagents; microchannels to and between these wells, includingmicrochannels containing electrophoretic separation matrices; valves tocontrol fluid movement; on-chip pumps such as electroosmotic,electrohydrodynamic, or electrokinetic pumps; and detection systemscomprising electrodes, as is more fully described below. The devices ofthe invention can be configured to manipulate one or multiple samples oranalytes.

The microfluidic devices of the invention are used to detect targetanalytes in samples. By “target analyte” or “analyte” or grammaticalequivalents herein is meant any molecule, compound or particle to bedetected. As outlined below, target analytes preferably bind to bindingligands, as is more fully described above. As will be appreciated bythose in the art, a large number of analytes may be detected using thepresent methods; basically, any target analyte for which a bindingligand, described herein, may be made may be detected using the methodsof the invention.

Suitable analytes include organic and inorganic molecules, includingbiomolecules. In a preferred embodiment, the analyte may be anenvironmental pollutant (including pesticides, insecticides, toxins,etc.); a chemical (including solvents, polymers, organic materials,etc.); therapeutic molecules (including therapeutic and abused drugs,antibiotics, etc.); biomolecules (including hormones, cytokines,proteins, lipids, carbohydrates, cellular membrane antigens andreceptors (neural, hormonal, nutrient, and cell surface receptors) ortheir ligands, etc); whole cells (including procaryotic (such aspathogenic bacteria) and eukaryotic cells, including mammalian tumorcells); viruses (including retroviruses, herpesviruses, adenoviruses,lentiviruses, etc.); and spores; etc. Particularly preferred analytesare environmental pollutants; nucleic acids; proteins (includingenzymes, antibodies, antigens, growth factors, cytokines, etc);therapeutic and abused drugs; cells; and viruses.

In a preferred embodiment, the target analyte is a nucleic acid. By“nucleic acid” or “oligonucleotide” or grammatical equivalents hereinmeans at least two nucleotides covalently linked together. A nucleicacid of the present invention will generally contain phosphodiesterbonds, although in some cases, as outlined below, nucleic acid analogsare included that may have alternate backbones, comprising, for example,phosphoramide (Beaucage et al., Tetrahedron 49(10):1925 (1993) andreferences therein; Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl etal., Eur. J. Biochem. 81:579 (1977); Letsinger et al., Nucl. Acids Res.14:3487 (1986); Sawai et al, Chem. Lett. 805 (1984), Letsinger et al.,J. Am. Chem. Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta26:141 91986)), phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437(1991); and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al.,J. Am. Chem. Soc. 111:2321 (1989), 0methylphosphoroamidite linkages (seeEckstein, Oligonucleotides and Analogues: A Practical Approach, OxfordUniversity Press), and peptide nucleic acid backbones and linkages (seeEgholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem. Int. Ed.Engl. 31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson et al.,Nature 380:207 (1996), all of which are incorporated by reference).Other analog nucleic acids include those with positive backbones (Denpcyet al., Proc. Natl. Acad. Sci. USA 92:6097 (1995); non-ionic backbones(U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423(1991); Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); Letsingeret al., Nucleoside & Nucleotide 13:1597 (1994); Chapters 2 and 3, ASCSymposium Series 580, “Carbohydrate Modifications in AntisenseResearch”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al.,Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J.Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996)) andnonribose backbones, including those described in U.S. Pat. Nos.5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,“Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghuiand P. Dan Cook. Nucleic acids containing one or more carbocyclic sugarsare also included within the definition of nucleic acids (see Jenkins etal., Chem. Soc. Rev. (1995) pp 169176). Several nucleic acid analogs aredescribed in Rawls, C & E News Jun. 2, 1997 page 35. All of thesereferences are hereby expressly incorporated by reference. Thesemodifications of the ribosephosphate backbone may be done to facilitatethe addition of electron transfer moieties, or to increase the stabilityand half-life of such molecules in physiological environments.

As will be appreciated by those in the art, all of these nucleic acidanalogs may find use in the present invention. In addition, mixtures ofnaturally occurring nucleic acids and analogs can be made; for example,at the site of conductive oligomer or electron transfer moietyattachment, an analog structure may be used. Alternatively, mixtures ofdifferent nucleic acid analogs, and mixtures of naturally occurringnucleic acids and analogs may be made.

Particularly preferred are peptide nucleic acids (PNA) which includespeptide nucleic acid analogs. These backbones are substantiallynon-ionic under neutral conditions, in contrast to the highly chargedphosphodiester backbone of naturally occurring nucleic acids. Thisresults in two advantages. First, the PNA backbone exhibits improvedhybridization kinetics. PNAs have larger changes in the meltingtemperature (Tm) for mismatched versus perfectly matched basepairs. DNAand RNA typically exhibit a 2-4° C. drop in Tm for an internal mismatch.With the non-ionic PNA backbone, the drop is closer to 7-9° C. Thisallows for better detection of mismatches. Similarly, due to theirnon-ionic nature, hybridization of the bases attached to these backbonesis relatively insensitive to salt concentration. This is particularlyadvantageous in the systems of the present invention, as a reduced salthybridization solution has a lower Faradaic current than a physiologicalsalt solution (in the range of 150 mM).

The nucleic acids may be single stranded or double stranded, asspecified, or contain portions of both double stranded or singlestranded sequence. The nucleic acid may be DNA, both genomic and cDNA,RNA or a hybrid, where the nucleic acid contains any combination ofdeoxyribo- and ribonucleotides, and any combination of bases, includinguracil, adenine, thymine, cytosine, guanine, inosine, xathaninehypoxathanine, isocytosine, isoguanine, etc. As used herein, the term“nucleoside” includes nucleotides and nucleoside and nucleotide analogs,and modified nucleosides such as amino modified nucleosides. Inaddition, “nucleoside” includes non-naturally occurring analogstructures. Thus for example the individual units of a peptide nucleicacid, each containing a base, are referred to herein as nucleosides.

In a preferred embodiment, the present invention provides methods ofdetecting target nucleic acids. By “target nucleic acid” or “targetsequence” or grammatical equivalents herein means a nucleic acidsequence on a single strand of nucleic acid. The target sequence may bea portion of a gene, a regulatory sequence, genomic DNA, cDNA, RNAincluding mRNA and rRNA, or others. It may be any length, with theunderstanding that longer sequences are more specific. In someembodiments, it may be desirable to fragment or cleave the samplenucleic acid into fragments of 100 to 10,000 basepairs, with fragmentsof roughly 500 basepairs being preferred in some embodiments. As will beappreciated by those in the art, the complementary target sequence maytake many forms. For example, it may be contained within a largernucleic acid sequence, i.e. all or part of a gene or mRNA, a restrictionfragment of a plasmid or genomic DNA, among others.

As is outlined more fully below, probes (including primers) are made tohybridize to target sequences to determine the presence or absence ofthe target sequence in a sample. Generally speaking, this term will beunderstood by those skilled in the art.

The target sequence may also be comprised of different target domains;for example, in “sandwich” type assays as outlined below, a first targetdomain of the sample target sequence may hybridize to a capture probe ora portion of capture extender probe, a second target domain mayhybridize to a portion of an amplifier probe, a label probe, or adifferent capture or capture extender probe, etc. In addition, thetarget domains may be adjacent (i.e. contiguous) or separated. Forexample, when ligation chain reaction (LCR) techniques are used, a firstprimer may hybridize to a first target domain and a second primer mayhybridize to a second target domain; either the domains are adjacent, orthey may be separated by one or more nucleotides, coupled with the useof a polymerase and dNTPs, as is more fully outlined below.

The terms “first” and “second” are not meant to confer an orientation ofthe sequences with respect to the 5′-3′ orientation of the targetsequence. For example, assuming a 5′-3′ orientation of the complementarytarget sequence, the first target domain may be located either 5′ to thesecond domain, or 3′ to the second domain.

In a preferred embodiment, the target analyte is a protein. As will beappreciated by those in the art, there are a large number of possibleproteinaceous target analytes that may be detected using the presentinvention. By “proteins” or grammatical equivalents herein is meantproteins, oligopeptides and peptides, derivatives and analogs, includingproteins containing non-naturally occurring amino acids and amino acidanalogs, and peptidomimetic structures. The side chains may be in eitherthe (R) or the (S) configuration. In a preferred embodiment, the aminoacids are in the (S) or L-configuration. As discussed below, when theprotein is used as a binding ligand, it may be desirable to utilizeprotein analogs to retard degradation by sample contaminants.

Suitable protein target analytes include, but are not limited to, (1)immunoglobulins, particularly IgEs, IgGs and IgMs, and particularlytherapeutically or diagnostically relevant antibodies, including but notlimited to, for example, antibodies to human albumin, apolipoproteins(including apolipoprotein E), human chorionic gonadotropin, cortisol,α-fetoprotein, thyroxin, thyroid stimulating hormone (TSH),antithrombin, antibodies to pharmaceuticals (including antieptilepticdrugs (phenyloin, primidone, carbariezepin, ethosuximide, valproic acid,and phenobarbitol), cardioactive drugs (digoxin, lidocaine,procainamide, and disopyramide), bronchodilators (theophylline),antibiotics (chloramphenicol, sulfonamides), antidepressants,immunosuppresants, abused drugs (amphetamine, methamphetamine,cannabinoids, cocaine and opiates) and antibodies to any number ofviruses (including orthomyxoviruses, (e.g. influenza virus),paramyxoviruses (e.g respiratory syncytial virus, mumps virus, measlesvirus), adenoviruses, rhinoviruses, coronaviruses, reoviruses,togaviruses (e.g. rubella virus), parvoviruses, poxviruses (e.g. variolavirus, vaccinia virus), enteroviruses (e.g. poliovirus, coxsackievirus);hepatitis viruses (including A, B and C), herpesviruses (e.g. Herpessimplex virus, varicella-zoster virus, cytomegalovirus, Epstein-Barrvirus), rotaviruses, Norwalk viruses, hantavirus, arenavirus,rhabdovirus (e.g. rabies virus), retroviruses (including HIV, HTLV-I and-II), papovaviruses (e.g. papillomavirus), polyomaviruses, andpicornaviruses, and the like), and bacteria (including a wide variety ofpathogenic and non-pathogenic prokaryotes of interest includingBacillus; Vibrio, e.g. V. cholerae; Escherichia, e.g. Enterotoxigenic E.coli, Shigella, e.g. S. dysenteriae; Salmonella, e.g. S. typhi;Mycobacterium e.g. M. tuberculosis, M. leprae; Clostridium, e.g. C.botulinum, C. tetani, C. difficile, C. perfringens; Cornyebacterium,e.g. C. diphtheriae; Streptococcus, S. pyogenes, S. pneumoniae;Staphylococcus, e.g. S. aureus; Haemophilus, e.g. H. influenzae;Neisseria, e.g. N. meningitidis, N. gonorrhoeae; Yersinia, e.g. G.lamblia Y. pestis, Pseudomonas, e.g. P. aeruginosa, P. putida;Chlamydia, e.g. C. trachomatis; Bordetella, e.g. B. pertussis;Treponema, e.g. T. palladium; and the like); (2) enzymes (and otherproteins), including but not limited to, enzymes used as indicators ofor treatment for heart disease, including creatine kinase, lactatedehydrogenase, aspartate amino transferase, troponin T, myoglobin,fibrinogen, cholesterol, triglycerides, thrombin, tissue plasminogenactivator (PA); pancreatic disease indicators including amylase, lipase,chymotrypsin and trypsin; liver function enzymes and proteins includingcholinesterase, bilirubin, and alkaline phosphotase; aldolase, prostaticacid phosphatase, terminal deoxynucleotidyl transferase, and bacterialand viral enzymes such as HIV protease; (3) hormones and cytokines (manyof, which serve as ligands for cellular receptors) such aserythropoietin (EPO), thrombopoietin (TPO), the interleukins (includingIL-1 through IL-17), insulin, insulin-like growth factors (includingIGF-1 and -2), epidermal growth factor (EGF), transforming growthfactors (including TGF-a and TGF-(3), human growth hormone, transferrin,epidermal growth factor (EGF), low density lipoprotein, high densitylipoprotein, leptin, VEGF, PDGF, ciliary neurotrophic factor, prolactin,adrenocorticotropic hormone (ACTH), calcitonin, human chorionicgonadotropin, cotrisol, estradiol, follicle stimulating hormone (FSH),thyroid-stimulating hormone (TSH), leutinzing hormone (LH), progeteroneand testosterone; and (4) other proteins (including a-fetoprotein,carcinoembryonic antigen CEA, cancer markers, etc.).

In addition, any of the biomolecules for which antibodies may bedetected may be detected directly as well; that is, detection of virusor bacterial cells, therapeutic and abused drugs, etc., may be donedirectly.

Suitable target analytes include carbohydrates, including but notlimited to, markers for breast cancer (CA15-3, CA 549, CA 27.29),mucin-like carcinoma associated antigen (MCA), ovarian cancer (CA125),pancreatic cancer (DE-PAN-2), prostate cancer (PSA), CEA, and colorectaland pancreatic cancer (CA 19, CA 50, CA242).

Suitable target analytes include metal ions, particularly heavy and/ortoxic metals, including but not limited to, aluminum, arsenic, cadmium,selenium, cobalt, copper, chromium, lead, silver and nickel.

These target analytes may be present in any number of different sampletypes, including, but not limited to, bodily fluids including blood,lymph, saliva, vaginal and anal secretions, urine, feces, perspirationand tears, and solid tissues, including liver, spleen, bone marrow,lung, muscle, brain, etc.

Accordingly, the present invention provides microfluidic devices for thedetection of target analytes comprising a solid substrate. The solidsubstrate can be made of a wide variety of materials and can beconfigured in a large number of ways, as is discussed herein and will beapparent to one of skill in the art. In addition, a single device may becomprises of more than one substrate; for example, there may be a“sample treatment” cassette that interfaces with a separate “detection”cassette; a raw sample is added to the sample treatment cassette and ismanipulated to prepare the sample for detection, which is removed fromthe sample treatment cassette and added to the detection cassette. Theremay be an additional functional cassette into which the device fits; forexample, a heating element which is placed in contact with the samplecassette to effect reactions such as PCR. In some cases, a portion ofthe substrate may be removable; for example, the sample cassette mayhave a detachable detection cassette, such that the entire samplecassette is not contacted with the detection apparatus. See for exampleU.S. Pat. No. 5,603,351 and PCT US96/17116, hereby incorporated byreference.

The composition of the solid substrate will depend on a variety offactors, including the techniques used to create the device, the use ofthe device, the composition of the sample, the analyte to be detected,the size of the wells and microchannels, the presence or absence ofelecronic components, etc. Generally, the devices of the inventionshould be easily sterilizable as well.

In a preferred embodiment, the solid substrate can be made from a widevariety of materials, including, but not limited to, silicon such assilicon wafers, silicon dioxide, silicon nitride, glass and fusedsilica, gallium arsenide, indium phosphide, aluminum, ceramics,polyimide, quartz, plastics, resins and polymers includingpolymethylmethacrylate, acrylics, polyethylene, polyethyleneterepthalate, polycarbonate, polystyrene and other styrene copolymers,polypropylene, polytetrafluoroethylene, superalloys, zircaloy, steel,gold, silver, copper, tungsten, molybdeumn, tantalum, KOVAR, KEVLAR,KAPTON, MYLAR, brass, sapphire, etc. High quality glasses such as highmelting borosilicate or fused silicas may be preferred for their UVtransmission properties when any of the sample manipulation stepsrequire light based technologies. In addition, as outlined herein,portions of the internal surfaces of the device may be coated with avariety of coatings as needed, to reduce nonspecific binding, to allowthe attachment of binding ligands, for biocompatibility, for flowresistance, etc.

The devices of the invention can be made in a variety of ways, as willbe appreciated by those in the art. See for example WO96/39260, directedto the formation of fluid-tight electrical conduits; U.S. Pat. No.5,747,169, directed to sealing; EP 0637996 131; EP 0637998 131;WO96/39260; WO97/16835; WO98/13683; WO97/16561; WO97/43629; WO96/39252;WO96/15576; WO96/15450; WO97/37755; and WO97/27324; and U.S. Pat. Nos.5,304,487; 5,071,531; 5,061,336; 5,747,169; 5,296,375; 5,110,745;5,587,128; 5,498,392; 5,643,738; 5,750,015; 5,726,026; 5,35,358;5,126,022; 5,770,029; 5,631,337; 5,569,364; 5,135,627; 5,632,876;5,593,838; 5,585,069; 5,637,469; 5,486,335; 5,755,942; 5,681,484; and5,603,351, all of which are hereby incorporated by reference. Suitablefabrication techniques again will depend on the choice of substrate, butpreferred methods include, but are not limited to, a variety ofmicromachining and microfabrication techniques, including filmdeposition processes such as spin coating, chemical vapor deposition,laser fabrication, photolithographic and other etching techniques usingeither wet chemical processes or plasma processes, embossing, injectionmolding and bonding techniques (see U.S. Pat. No. 5,747,169, herebyincorporated by reference). In addition, there are printing techniquesfor the creation of desired fluid guiding pathways; that is, patterns ofprinted material can permit directional fluid transport. Thus, thebuild-up of “ink” can serve to define a flow channel. In addition, theuse of different “inks” or “pastes” can allow different portions of thepathways having different flow properties. For example, materials can beused to change solute/solvent RF values (the ratio of the distance movedby a particular solute to that moved by a solvent front). For example,printed fluid guiding pathways can be manufactured with a printed layeror layers comprised of two different materials, providing differentrates of fluid transport. Multi-material fluid guiding pathways can beused when it is desirable to modify retention times of reagents in fluidguiding pathways. Furthermore, printed fluid guiding pathways can alsoprovide regions containing reagent substances, by including the reagentsin the “inks” or by a subsequent printing step. See for example U.S.Pat. No. 5,795,453, herein incorporated by reference in its entirety.

In a preferred embodiment, the solid substrate is configured forhandling a single sample that may contain a plurality of targetanalytes. That is, a single sample is added to the device and the samplemay either be aliquoted for parallel processing for detection of theanalytes or the sample may be processed serially, with individualtargets being detected in a serial fashion. In addition, samples may beremoved periodically or from different locations for in line sampling.

In a preferred embodiment, the solid substrate is configured forhandling multiple samples, each of which may contain one or more targetanalytes. In general, in this embodiment, each sample is handledindividually; that is, the manipulations and analyses are done inparallel, with preferably no contact or contamination between them.Alternatively, there may be some steps in common; for example, it may bedesirable to process different samples separately but detect all of thetarget analytes on a single detection electrode, as described below.

In addition, it should be understood that while most of the discussionherein is directed to the use of planar substrates with microchannelsand wells, other geometries can be used as well. For example, two ormore planar substrates can be stacked to produce a three dimensionaldevice, that can contain microchannels flowing within one plane orbetween planes; similarly, wells may span two or more substrates toallow for larger sample volumes. Thus for example, both sides of asubstrate can be etched to contain microchannels; see for example U.S.Pat. Nos. 5,603,351 and 5,681,484, both of which are hereby incorporatedby reference.

Thus, the devices of the invention include at least one microchannel orflow channel that allows the flow of sample from the sample inlet portto the other components or modules of the system. The collection ofmicrochannels and wells is sometimes referred to in the art as a“mesoscale flow system”. As will be appreciated by those in the art, theflow channels may be configured in a wide variety of ways, depending onthe use of the channel. For example, a single flow channel starting atthe sample inlet port may be separated into a variety of smallerchannels, such that the original sample is divided into discretesubsamples for parallel processing or analysis. Alternatively, severalflow channels from different modules, for example the sample inlet portand a reagent storage module may feed together into a mixing chamber ora reaction chamber. As will be appreciated by those in the art, thereare a large number of possible configurations; what is important is thatthe flow channels allow the movement of sample and reagents from onepart of the device to another. For example, the path lengths of the flowchannels may be altered as needed; for example, when mixing and timedreactions are required, longer and sometimes tortuous flow channels canbe used.

In general, the microfluidic devices of the invention are generallyreferred to as “mesoscale” devices. The devices herein are typicallydesigned on a scale suitable to analyze microvolumes, although in someembodiments large samples (e.g. cc's of sample) may be reduced in thedevice to a small volume for subsequent analysis. That is, “mesoscale”as used herein refers to chambers and microchannels that havecross-sectional dimensions on the order of 0.1 μm to 500 μm. Themesoscale flow channels and wells have preferred depths on the order of0.1 μm to 100 μm, typically 2-50 μm. The channels have preferred widthson the order of 2.0 to 500 μm, more preferably 3-100 μm. For manyapplications, channels of 5-50 μm are useful. However, for manyapplications, larger dimensions on the scale of millimeters may be used.Similarly, chambers (sometimes also referred to herein as “wells”) inthe substrates often will have larger dimensions, on the scale of a fewmillimeters.

In addition to the flow channel system, the devices of the invention areconfigured to include one or more of a variety of components, hereinreferred to as “modules”, that will be present on any given devicedepending on its use. These modules include, but are not limited to:sample inlet ports; sample introduction or collection modules; cellhandling modules (for example, for cell lysis, cell removal, cellconcentration, cell separation or capture, cell growth, etc.);separation modules, for example, for electrophoresis, dielectrophoresis,gel filtration, ion exchange/affinity chromatography (capture andrelease) etc.; reaction modules for chemical or biological alteration ofthe sample, including amplification of the target analyte (for example,when the target analyte is nucleic acid, amplification techniques areuseful, including, but not limited to polymerase chain reaction (PCR),ligase chain reaction (LCR), strand displacement amplification (SDA),and nucleic acid sequence based amplification (NASBA)), chemical,physical or enzymatic cleavage or alteration of the target analyte, orchemical modification of the target; fluid pumps; fluid valves; thermalmodules for heating and cooling; storage modules for assay reagents;mixing chambers; and detection modules.

In a preferred embodiment, the devices of the invention, include atleast one sample inlet port for the introduction of the sample to thedevice. This may be part of or separate from a sample introduction orcollection module; that is, the sample may be directly fed in from thesample inlet port to a separation chamber, or it may be pretreated in asample collection well or chamber.

In a preferred embodiment, the devices of the invention include a samplecollection module, which can be used to concentrate or enrich the sampleif required; for example, see U.S. Pat. No. 5,770,029, including thediscussion of enrichment channels and enrichment means.

In a preferred embodiment, the devices of the invention include a cellhandling module. This is of particular use when the sample comprisescells that either contain the target analyte or that must be removed inorder to detect the target analyte. Thus, for example, the detection ofparticular antibodies in blood can require the removal of the bloodcells for efficient analysis, or the cells (and/or nucleus) must belysed prior to detection. In this context, “cells” include eukaryoticand prokaryotic cells, and viral particles that may require treatmentprior to analysis, such as the release of nucleic acid from a viralparticle prior to detection of target sequences. In addition, cellhandling modules may also utilize a downstream means for determining thepresence or absence of cells, Suitable cell handling modules include,but are not limited to, cell lysis modules, cell removal modules, cellconcentration modules, and cell separation or capture modules. Inaddition, as for all the modules of the invention, the cell handlingmodule is in fluid communication via a flow channel with at least oneother module of the invention.

In a preferred embodiment, the cell handling module includes a celllysis module. As is known in the art, cells may be lysed in a variety ofways, depending on the cell type. In one embodiment, as described in EP0 637 998 B1 and U.S. Pat. No. 5,635,358, hereby incorporated byreference, the cell lysis module may comprise cell membrane piercingprotrusions that extend from a surface of the cell handling module. Asfluid is forced through the device, the cells are ruptured. Similarly,this may be accomplished using sharp edged particles trapped within thecell handling region. Alternatively, the cell lysis module can comprisea region of restricted cross-sectional dimension, which results in celllysis upon pressure.

In a preferred embodiment, the cell lysis module comprises a cell lysingagent, such as guanidium chloride, chaotropic salts, enzymes such aslysozymes, etc. In some embodiments, for example for blood cells, asimple dilution with water or buffer can result in hypotonic lysis. Thelysis agent may be solution form, stored within the cell lysis module orin a storage module and pumped into the lysis module. Alternatively, thelysis agent may be in solid form, that is taken up in solution uponintroduction of the sample.

The cell lysis module may also include, either internally or externally,a filtering module for the removal of cellular debris as needed. Thisfilter may be microfabricated between the cell lysis module and thesubsequent module to enable the removal of the lysed cell membrane andother cellular debris components; examples of suitable filters are shownin EP 0 637 998 B1, incorporated by reference.

In a preferred embodiment, the cell handling module includes a cellseparation or capture module. This embodiment utilizes a cell captureregion comprising binding sites capable of reversibly binding a cellsurface molecule to enable the selective isolation (or removal) of aparticular type of cell from the sample population, for example, whiteblood cells for the analysis of chromosomal nucleic acid, or subsets ofwhite blood cells. These binding moieties may be immobilized either onthe surface of the module or on a particle trapped within the module(i.e. a bead) by physical absorption or by covalent attachment. Suitablebinding moieties will depend on the cell type to be isolated or removed,and generally includes antibodies and other binding ligands, such asligands for cell surface receptors, etc.

Thus, a particular cell type may be removed from a sample prior tofurther handling, or the assay is designed to specifically bind thedesired cell type, wash away the non-desirable cell types, followed byeither release of the bound cells by the addition of reagents orsolvents, physical removal (i.e. higher flow rates or pressures), oreven in situ lysis.

Alternatively, a cellular “sieve” can be used to separate cells on thebasis of size. This can be done in a variety of ways, includingprotrusions from the surface that allow size exclusion, a series ofnarrowing channels, a weir, or a diafiltration type setup.

In a preferred embodiment, the cell handling module includes a cellremoval module. This may be used when the sample contains cells that arenot required in the assay or are undesirable. Generally, cell removalwill be done on the basis of size exclusion as for “sieving”, above,with channels exiting the cell handling module that are too small forthe cells.

In a preferred embodiment, the cell handling module includes a cellconcentration module. As will be appreciated by those in the art, thisis done using “sieving” methods, for example to concentrate the cellsfrom a large volume of sample fluid prior to lysis.

In a preferred embodiment, the devices of the invention include aseparation module. Separation in this context means that at least onecomponent of the sample is separated from other components of thesample. This can comprise the separation or isolation of the targetanalyte, or the removal of contaminants that interfere with the analysisof the target analyte, depending on the assay.

In a preferred embodiment, the separation module includeschromatographic-type separation media such as absorptive phasematerials, including, but not limited to reverse phase materials (e.g.C₈ or C₁₈ coated particles, etc.), ion-exchange materials, affinitychromatography materials such as binding ligands, etc. See U.S. Pat. No.5,770,029, herein incorporated by reference.

In a preferred embodiment, the separation module utilizes bindingligands, as is generally outlined herein for cell separation or analytedetection. In this embodiment, binding ligands are immobilized (again,either by physical absorption or covalent attachment, described below)within the separation module (again, either on the internal surface ofthe module, on a particle such as a bead, filament or capillary trappedwithin the module, for example through the use of a frit). Suitablebinding moieties will depend on the sample component to be isolated orremoved. By “binding ligand” or grammatical equivalents herein is meanta compound that is used to bind a component of the sample, either acontaminant (for removal) or the target analyte (for enrichment). Insome embodiments, as outlined below, the binding ligand is used to probefor the presence of the target analyte, and that will bind to theanalyte.

As will be appreciated by those in the art, the composition of thebinding ligand will depend on the sample component to be separated.Binding ligands for a wide variety of analytes are known or can bereadily found using known techniques. For example, when the component isa protein, the binding ligands include proteins (particularly includingantibodies or fragments thereof (FAbs, etc.)) or small molecules. Whenthe sample component is a metal ion, the binding ligand generallycomprises traditional metal ion ligands or chelators. Preferred bindingligand proteins include peptides. For example, when the component is anenzyme, suitable binding ligands include substrates and inhibitors.Antigen-antibody pairs, receptor-ligands, and carbohydrates and theirbinding partners are also suitable component-binding ligand pairs. Thebinding ligand may be nucleic acid, when nucleic acid binding proteinsare the targets; alternatively, as is generally described in U.S. Pat.Nos. 5,270,163, 5,475,096, 5,567,588, 5,595,877, 5,637,459, 5,683,867,5,705,337, and related patents, hereby incorporated by reference,nucleic acid “aptomers” can be developed for binding to virtually anytarget analyte. Similarly, there is a wide body of literature relatingto the development of binding partners based on combinatorial chemistrymethods. In this embodiment, when the binding ligand is a nucleic acid,preferred compositions and techniques are outlined in PCT US97/20014,hereby incorporated by reference.

In a preferred embodiment, the binding of the sample component to thebinding ligand is specific, and the binding ligand is part of a bindingpair. By “specifically bind” herein is meant that the ligand binds thecomponent, for example the target analyte, with specificity sufficientto differentiate between the analyte and other components orcontaminants of the test sample. The binding should be sufficient toremain bound under the conditions of the separation step or assay,including wash steps to remove non-specific binding. In someembodiments, for example in the detection of certain biomolecules, thedisassociation constants of the analyte to the binding ligand will beless than about 10⁻⁴-10⁻⁶ M⁻¹, with less than about 10⁻⁵ to 10⁻⁹ M⁻¹being preferred and less than about 10⁻⁷-10⁻⁹ M⁻¹ being particularlypreferred.

As will be appreciated by those in the art, the composition of thebinding ligand will depend on the composition of the target analyte.Binding ligands to a wide variety of analytes are known or can bereadily found using known techniques. For example, when the analyte is asingle-stranded nucleic acid, the binding ligand is generally asubstantially complementary nucleic acid. Similarly the analyte may be anucleic acid binding protein and the capture binding ligand is either asingle-stranded or double-stranded nucleic acid; alternatively, thebinding ligand may be a nucleic acid binding protein when the analyte isa single or double-stranded nucleic acid. When the analyte is a protein,the binding ligands include proteins or small molecules. Preferredbinding ligand proteins include peptides. For example, when the analyteis an enzyme, suitable binding ligands include substrates, inhibitors,and other proteins that bind the enzyme, i.e. components of amulti-enzyme (or protein) complex. As will be appreciated by those inthe art, any two molecules that will associate, preferably specifically,may be used, either as the analyte or the binding ligand. Suitableanalyte/binding ligand pairs include, but are not limited to,antibodies/antigens, receptors/ligand, proteins/nucleic acids; nucleicacids/nucleic acids, enzymes/substrates and/or inhibitors, carbohydrates(including glycoproteins and glycolipids)/lectins, carbohydrates andother binding partners, proteins/proteins; and protein/small molecules.These may be wild type or derivative sequences. In a preferredembodiment, the binding ligands are portions (particularly theextracellular portions) of cell surface receptors that are known tomultimerize, such as the growth hormone receptor, glucose transporters(particularly GLUT4 receptor), transferrin receptor, epidermal growthfactor receptor, low density lipoprotein receptor, high densitylipoprotein receptor, leptin receptor, interleukin receptors includingIL1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12,IL-13, IL-15 and IL-17 receptors, VEGF receptor, PDGF receptor, EPOreceptor, TPO receptor, ciliary neurotrophic factor receptor, prolactinreceptor, and T-cell receptors.

When the sample component bound by the binding ligand is the targetanalyte, it may be released for detection purposes if necessary, usingany number of known techniques, depending on the strength of the bindinginteraction, including changes in pH, salt concentration, temperature,etc. or the addition of competing ligands, detergents, chaotropicagents, organic compounds, or solvents, etc.

In some embodiments, preferential binding of molecules to surfaces canbe achieved using coating agents or buffer conditions; for example, DNAand RNA may be differentially bound to glass surfaces depending on theconditions.

In a preferred embodiment, the separation module includes anelectrophoresis module, as is generally described in U.S. Pat. Nos.5,770,029; 5,126,022; 5,631,337; 5,569,364; 5,750,015, and 5,135,627,all of which are hereby incorporated by reference. In electrophoresis,molecules are primarily separated by different electrophoreticmobilities caused by their different molecular size, shape and/orcharge. Microcapillary tubes have recently been used for use inmicrocapillary gel electrophoresis (high performance capillaryelectrophoresis (HPCE)). One advantage of HPCE is that the heatresulting from the applied electric field is efficiently disappated dueto the high surface area, thus allowing fast separation. Theelectrophoresis module serves to separate sample components by theapplication of an electric field, with the movement of the samplecomponents being due either to their charge or, depending on the surfacechemistry of the microchannel, bulk fluid flow as a result ofelectroosmotic flow (EOF).

As will be appreciated by those in the art, the electrophoresis modulecan take on a variety of forms, and generally comprises anelectrophoretic microchannel and associated electrodes to apply anelectric field to the electrophoretic microchannel. Waste fluid outletsand fluid reservoirs are present as required.

The electrodes comprise pairs of electrodes, either a single pair, or,as described in U.S. Pat. Nos. 5,126,022 and 5,750,015, a plurality ofpairs. Single pairs generally have one electrode at each end of theelectrophoretic pathway. Multiple electrode pairs may be used toprecisely control the movement of sample components, such that thesample components may be continuously subjected to a plurality ofelectric fields either simultaneously or sequentially.

In a preferred embodiment, electrophoretic gel media may also be used.By varying the pore size of the media, employing two or more gel mediaof different porosity, and/or providing a pore size gradient, separationof sample components can be maximized. Gel media for separation based onsize are known, and include, but are not limited to, polyacrylamide andagarose. One preferred electrophoretic separation matrix is described inU.S. Pat. No. 5,135,627, hereby incorporated by reference, thatdescribes the use of “mosaic matrix”, formed by polymerizing adispersion of microdomains (“dispersoids”) and a polymeric matrix. Thisallows enhanced separation of target analytes, particularly nucleicacids. Similarly, U.S. Pat. No. 5,569,364, hereby incorporated byreference, describes separation media for electrophoresis comprisingsubmicron to above-micron sized crosslinked gel particles that find usein microfluidic systems. U.S. Pat. No. 5,631,337, hereby incorporated byreference, describes the use of thermoreversible hydrogels comprisingpolyacrylamide backbones with N-substituents that serve to providehydrogen bonding groups for improved electrophoretic separation. Seealso U.S. Pat. Nos. 5,061,336 and 5,071,531, directed to methods ofcasting gels in capillary tubes.

In a preferred embodiment, the devices of the invention include areaction module. This can include either physical, chemical orbiological alteration of one or more sample components. Alternatively,it may include a reaction module wherein the target analyte alters asecond moiety that can then be detected; for example, if the targetanalyte is an enzyme, the reaction chamber may comprise an enzymesubstrate that upon modification by the target analyte, can then bedetected. In this embodiment, the reaction module may contain thenecessary reagents, or they may be stored in a storage module and pumpedas outlined herein to the reaction module as needed.

In a preferred embodiment, the reaction module includes a chamber forthe chemical modification of all or part of the sample. For example,chemical cleavage of sample components (CNBr cleavage of proteins, etc.)or chemical cross-linking can be done. PCT US97/07880, herebyincorporated by lists a large number of possible chemical reactions thatcan be done in the devices of the invention, including amide formation,acylation, alkylation, reductive amination, Mitsunobu, Diels Alder andMannich reactions, Suzuki and Stifle coupling, chemical labeling, etc.Similarly, U.S. Pat. Nos. 5,616,464 and 5,767,259 describe a variationof LCR that utilizes a “chemical ligation” of sorts. In this embodiment,similar to LCR, a pair of primers are utilized, wherein the first primeris substantially complementary to a first domain of the target and thesecond primer is substantially complementary t an adjacent second domainof the target (although, as for LCR, if a “gap” exists, a polymerase anddNTPs may be added to “fill in” the gap). Each primer has a portion thatacts as a “side chain” that does not bind the target sequence and actsas one half of a stem structure that interacts noncovalently throughhydrogen bonding, salt bridges, van der Waal's forces, etc. Preferredembodiments utilize substantially complementary nucleic acids as theside chains. Thus, upon hybridization of the primers to the targetsequence, the side chains of the primers are brought into spatialproximity, and, if the side chains comprise nucleic acids as well, canalso form side chain hybridization complexes. At least one of the sidechains of the primers comprises an activatable cross-linking agent,generally covalently attached to the side chain, that upon activation,results in a chemical cross-link or chemical ligation. The activatiblegroup may comprise any moiety that will allo cross-linking of the sidechains, and include groups activated chemically, photonically andthermally, with photoactivatable groups being preferred. In someembodiments a single activatable group on on of the side chains isenough to result in cross-linking via interaction to a functional groupon the other side chain; in alternate embodiments, activatable groupsare required on each side chain. In addition, the reaction chamber maycontain chemical moieties for the protection or deprotection of certainfunctional groups, such as thiols or amines.

In a preferred embodiment, the reaction module includes a chamber forthe biological alteration of all or part of the sample. For example,enzymatic processes including nucleic acid amplification, hydrolysis ofsample components or the hydrolysis of substrates by a target enzyme,the addition or removal of detectable labels, the addition or removal ofphosphate groups, etc.

In a preferred embodiment, the target analyte is a nucleic acid and thebiological reaction chamber allows amplification of the target nucleicacid. Suitable amplification techniques include, both targetamplification and probe amplification, including, but not limited to,polymerase chain reaction (PCR), ligase chain reaction (LCR), stranddisplacement amplification (SDA), self-sustained sequence replication(3SR), QB replicase amplification (QBR), repair chain reaction (RCR),cycling probe technology or reaction (CPT or CPR), and nucleic acidsequence based amplification (NASBA). Techniques utilizing these methodsand the detection modules of the invention are described in POTUS99/01705, herein incorporated by reference in its entirety. In thisembodiment, the reaction reagents generally comprise at least one enzyme(generally polymerase), primers, and nucleoside triphosphates as needed.

General techniques for nucleic acid amplification are discussed below.In most cases, double stranded target nucleic acids are denatured torender them single stranded so as to permit hybridization of the primersand other probes of the invention. A preferred embodiment utilizes athermal step, generally by raising the temperature of the reaction toabout 95° C., although pH changes and other techniques such as the useof extra probes or nucleic acid binding proteins may also be used.

A probe nucleic acid (also referred to herein as a primer nucleic acid)is then contacted to the target sequence to form a hybridizationcomplex. By “primer nucleic acid” herein is meant a probe nucleic acidthat will hybridize to some portion, i.e. a domain, of the targetsequence. Probes of the present invention are designed to becomplementary to a target sequence (either the target sequence of thesample or to other probe sequences, as is described below), such thathybridization of the target sequence and the probes of the presentinvention occurs. As outlined below, this complementarity need not beperfect; there may be any number of base pair mismatches which willinterfere with hybridization between the target sequence and the singlestranded nucleic acids of the present invention. However, if the numberof mutations is so great that no hybridization can occur under even theleast stringent of hybridization conditions, the sequence is not acomplementary target sequence. Thus, by “substantially complementary”herein is meant that the probes are sufficiently complementary to thetarget sequences to hybridize under normal reaction conditions.

A variety of hybridization conditions may be used in the presentinvention, including high, moderate and low stringency conditions; seefor example Maniatis et al., Molecular Cloning: A Laboratory Manual, 2dEdition, 1989, and Short Protocols in Molecular Biology, ed. Ausubel, etal, hereby incorporated by reference. Stringent conditions aresequence-dependent and will be different in different circumstances.Longer sequences hybridize specifically at higher temperatures. Anextensive guide to the hybridization of nucleic acids is found inTijssen, Techniques in Biochemistry and Molecular Biology—Hybridizationwith Nucleic Acid Probes, “Overview of principles of hybridization andthe strategy of nucleic acid assays” (1993). Generally, stringentconditions are selected to be about 5-10° C. lower than the thermalmelting point (Tm) for the specific sequence at a defined ionic strengthpH. The Tm is the temperature (under defined ionic strength, pH andnucleic acid concentration) at which 50% of the probes complementary tothe target hybridize to the target sequence at equilibrium (as thetarget sequences are present in excess, at Tm, 50% of the probes areoccupied at equilibrium). Stringent conditions will be those in whichthe salt concentration is less than about 1.0 sodium ion, typicallyabout 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0to 8.3 and the temperature is at least about 30° C. for short probes(e.g. 10 to 50 nucleotides) and at least about 60° C. for long probes(e.g. greater than 50 nucleotides). Stringent conditions may also beachieved with the addition of destabilizing agents such as formamide.The hybridization conditions may also vary when a non-ionic backbone,i.e. PNA is used, as is known in the art. In addition, crosslinkingagents may be added after target binding to cross-link, i.e. covalentlyattach, the two strands of the hybridization complex.

Thus, the assays are generally run under stringency conditions whichallows formation of the hybridization complex only in the presence oftarget. Stringency can be controlled by altering a step parameter thatis a thermodynamic variable, including, but not limited to, temperature,formamide concentration, salt concentration, chaotropic saltconcentration pH, organic solvent concentration, etc.

These parameters may also be used to control non-specific binding, as isgenerally outlined in U.S. Pat. No. 5,681,697. Thus it may be desirableto perform certain steps at higher stringency conditions to reducenon-specific binding.

The size of the primer nucleic acid may vary, as will be appreciated bythose in the art, in general varying from 5 to 500 nucleotides inlength, with primers of between 10 and 100 being preferred, between 15and 50 being particularly preferred, and from 10 to 35 being especiallypreferred, depending on the use and amplification technique.

In addition, the different amplification techniques may have furtherrequirements of the primers, as is more fully described below.

Once the hybridization complex between the primer and the targetsequence has been formed, an enzyme, sometimes termed an “amplificationenzyme”, is used to modify the primer. As for all the methods outlinedherein, the enzymes may be added at any point during the assay, eitherprior to, during, or after the addition of the primers. Theidentification of the enzyme will depend on the amplification techniqueused, as is more fully outlined below. Similarly, the modification willdepend on the amplification technique, as outlined below, althoughgenerally the first step of all the reactions herein is an extension ofthe primer, that is, nucleotides are added to the primer to extend itslength.

Once the enzyme has, modified the primer to form a modified primer, thehybridization complex is disassociated. Generally, the amplificationsteps are repeated for a period of time to allow a number of cycles,depending on the number of copies of the original target sequence andthe sensitivity of detection, with cycles ranging from 1 to thousands,with from 10 to 100 cycles being preferred and from 20 to 50 cyclesbeing especially preferred.

After a suitable time or amplification, the modified primer is moved toa detection module and incorporated into an assay complex, as is morefully outlined below. The assay complex is covalently attached to anelectrode, and comprises at least one electron transfer moiety (ETM),described below. Electron transfer between the ETM and the electrode isthen detected to indicate the presence or absence of the original targetsequence, as described below.

In a preferred embodiment, the amplification is target amplification.Target amplification involves the amplification (replication) of thetarget sequence to be detected, such that the number of copies of thetarget sequence is increased. Suitable target amplification techniquesinclude, but are not limited to, the polymerase chain reaction (PCR),strand displacement amplification (SDA), and nucleic acid sequence basedamplification (NASBA).

In a preferred embodiment, the target amplification technique is PCR.The polymerase chain reaction (PCR) is widely used and described, andinvolve the use of primer extension combined with thermal cycling toamplify a target sequence; see U.S. Pat. Nos. 4,683,195 and 4,683,202,and FOR Essential Data, J. W. Wiley & sons, Ed. C. R. Newton, 1995, allof which are incorporated by reference. In addition, there are a numberof variations of PCR which also find use in the invention, including“quantitative competitive FOR” or “QC-PCR”, “arbitrarily primed PCR” or“AP-PCR”, “immuno-PCR”, “Alu-PCR”, “PCR single strand conformationalpolymorphism” or “PCR-SSCP”, “reverse transcriptase PCR” or “RT-PCR”,“biotin capture PCR”, “vectorette PCR”. “panhandle PCR”, and “PCR selectcDNA subtration”, among others.

In general, PCR may be briefly described as follows. A double strandedtarget nucleic acid is denatured, generally by raising the temperature,and then cooled in the presence of an excess of a PCR primer, which thenhybridizes to the first target strand. A DNA polymerase then acts toextend the primer, resulting in the synthesis of a new strand forming ahybridization complex. The sample is then heated again, to disassociatethe hybridization complex, and the process is repeated. By using asecond PCR primer for the complementary target strand, rapid andexponential amplification occurs. Thus FOR steps are denaturation,annealing and extension. The particulars of PCR are well known, andinclude the use of a thermostabile polymerase such as Taq I polymeraseand thermal cycling.

Accordingly, the PCR reaction requires at least one PCR primer and apolymerase. Mesoscale PCR devices are described in U.S. Pat. Nos.5,498,392 and 5,587,128, and WO 97/16561, incorporated by reference.

In a preferred embodiment, the target amplification technique is SDA.Strand displacement amplification (SDA) is generally described in Walkeret al., in Molecular Methods for Virus Detection, Academic Press, Inc.,1995, and U.S. Pat. Nos. 5,455,166 and 5,130,238, all of which arehereby expressly incorporated by reference in their entirety.

In general, SDA may be described as follows. A single stranded targetnucleic acid, usually a DNA target sequence, is contacted with an SDAprimer. An “SDA primer” generally has a length of 25-100 nucleotides,with SDA primers of approximately 35 nucleotides being preferred. An SDAprimer is substantially complementary to a region at the 3′ end of thetarget sequence, and the primer has a sequence at its 5′ end (outside ofthe region that is complementary to the target) that is a recognitionsequence for a restriction endonuclease, sometimes referred to herein asa “nicking enzyme” or a “nicking endonuclease”, as outlined below. TheSDA primer then hybridizes to the target sequence. The SDA reactionmixture also contains a polymerase (an “SDA polymerase”, as outlinedbelow) and a mixture of all four deoxynucleoside-triphosphates (alsocalled deoxynucleotides or dNTPs, i.e. dATP, dTTP, dCTP and dGTP), atleast one species of which is a substituted or modified dNTP; thus, theSDA primer is modified, i.e. extended, to form a modified primer,sometimes referred to herein as a “newly synthesized strand”. Thesubstituted dNTP is modified such that it will inhibit cleavage in thestrand containing the substituted dNTP but will not inhibit cleavage onthe other strand. Examples of suitable substituted dNTPs include, butare not limited, 2′deoxyadenosine 5′-O-(1-thiotriphosphate),5-methyldeoxycytidine 5′-triphosphate, 2′-deoxyuridine 5′-triphosphate,adn 7-deaza-2′-deoxyguanosine 5′-triphosphate. In addition, thesubstitution of the dNTP may occur after incorporation into a newlysynthesized strand; for example, a methylase may be used to add methylgroups to the synthesized strand. In addition, if all the nucleotidesare substituted, the polymerase may have 3′ exonuclease activity.However, if less than all the nucleotides are substituted, thepolymerase preferably lacks 5′→′3′ exonuclease activity.

As will be appreciated by those in the art, the recognitionsitelendonuclease pair can be any of a wide variety of knowncombinations. The endonuclease is chosen to cleave a strand either atthe recognition site, or either 3′ or 5′ to it, without cleaving thecomplementary sequence, either because the enzyme only cleaves onestrand or because of the incorporation of the substituted nucleotides.Suitable recognition site/endonuclease pairs are well known in the art;suitable endonucleases include, but are not limited to, HincII, HindIII,AvaI, Fnu4HI, TthIIII, NclI, BstXI, BamI, etc. A chart depictingsuitable enzymes, and their corresponding recognition sites and themodified dNTP to use is found in U.S. Pat. No. 5,455,166, herebyexpressly incorporated by reference.

Once nicked, a polymerase (an “SDA polymerase”) is used to extend thenewly nicked strand, 5′→3′, thereby creating another newly synthesizedstrand. The polymerase chosen should be able to intiate 5′→3′polymerization at a nick site, should also displace the polymerizedstrand downstream from the nick, and should lack 5′→3′ exonucleaseactivity (this may be additionally accomplished by the addition of ablocking agent). Thus, suitable polymerases in SDA include, but are notlimited to, the Klenow fragment of DNA polymerase I, SEQUENASE 1.0 andSEQUENASE 2.0 (U.S. Biochemical), T5 DNA polymerase and Phi29 DNApolymerase.

Accordingly, the SDA reaction requires, in no particular order, an SDAprimer, an SDA polymerase, a nicking endonuclease, and dNTPs, at leastone species of which is modified.

In general, SDA does not require thermocycling. The temperature of thereaction is generally set to be high enough to prevent non-specifichybridization but low enough to allow specific hybridization; this isgenerally from about 37° C. to about 42° C., depending on the enzymes.

In a preferred embodiment, as for most of the amplification techniquesdescribed herein, a second amplification reaction can be done using thecomplementary target sequence, resulting in a substantial increase inamplification during a set period of time. That is, a second primernucleic acid is hybridized to a second target sequence, that issubstantially complementary to the first target sequence, to form asecond hybridization complex. The addition of the enzyme, followed bydisassociation of the second hybridization complex, results in thegeneration of a number of newly synthesized second strands.

In this way, a number of target molecules are made, and transferred to adetection module, described below. As is more fully outlined below,these reactions (that is, the products of these reactions) can bedetected in a number of ways. In general, either direct or indirectdetection of the target products can be done. “Direct” detection as usedin this context, as for the other amplification strategies outlinedherein, requires the incorporation of a label, in this case an electrontransfer moiety (ETM), into the target sequence, with detectionproceeding according to either “mechanism-1” or “mechanism-2”, outlinedbelow. In this embodiment, the ETM(s) may be incorporated in three ways;(1) the primers comprise the ETM(s), for example attached to the base, aribose, a phosphate, or to analogous structures in a nucleic acidanalog; (2) modified nucleosides are used that are modified at eitherthe base or the ribose (or to analogous structures in a nucleic acidanalog) with the ETM(s); these ETM modified nucleosides are thenconverted to the triphosphate form and are incorporated into the newlysynthesized strand by a polymerase; or (3) a “tail” of ETMs can beadded, as outlined below. Either of these methods result in a newlysynthesized strand that comprises ETMs, that can be directly detected asoutlined below.

Alternatively, indirect detection proceeds as a sandwich assay, with thenewly synthesized strands containing few or no ETMs. Detection thenproceeds via the use of label probes that comprise the ETM(s); theselabel probes will hybridize either directly to the newly synthesizedstrand or to intermediate probes such as amplification probes, as ismore fully outlined below. In this case, it is the ETMs on the labelprobes that are used for detection as outlined below.

In a preferred embodiment, the target amplification technique is nucleicacid sequence based amplification (NASBA). NASBA is generally describedin U.S. Pat. No. 5,409,818 and “Profiting from Gene-based Diagnostics”,CTB International Publishing Inc., N.J., 1996, both of which areexpressly incorporated by reference in their entirety.

In general, NASBA may be described as follows. A single stranded targetnucleic acid, usually an RNA target sequence (sometimes referred toherein as “the first target sequence” or “the first template”), iscontacted with a first NASBA primer. A “NASBA primer” generally has alength of 25100 nucleotides, with NASBA primers of approximately 50-75nucleotides being preferred. The first NASBA primer is preferably a DNAprimer that has at its 3′ end a sequence that is substantiallycomplementary to the Tend of the first template. The first NASBA primerhas an RNA polymerase promoter at its Fend. The first NASBA primer isthen hybridized to the first template to form a first hybridizationcomplex. The NASBA reaction mixture also includes a reversetranscriptase enzyme (an “NASBA reverse transcriptase”) and a mixture ofthe four dNTPs, such that the first NASBA primer is modified, i.e.extended, to form a modified first primer, comprising a hybridizationcomplex of RNA (the first template) and DNA (the newly synthesizedstrand).

By “reverse transcriptase” or “RNA-directed DNA polymerase” herein ismeant an enzyme capable of synthesizing DNA from a DNA primer and an RNAtemplate. Suitable RNA-directed DNA polymerases include, but are notlimited to, avian myloblastosis virus reverse transcriptase (“AMV RT”)and the Moloney murine leukemia virus RT.

In addition to the components listed above, the NASBA reaction alsoincludes an RNA degrading enzyme, also sometimes referred to herein as aribonuclease, that will hydrolyze RNA of an RNA:DNA hybrid withouthydrolyzing single- or double-stranded RNA or DNA. Suitableribonucleases include, but are not limited to, RNase H from E. coli andcalf thymus.

The ribonuclease degrades the first RNA template in the hybridizationcomplex, resulting in a disassociation of the hybridization complexleaving a first single stranded newly synthesized DNA strand, sometimesreferred to herein as “the second template”.

In addition, the NASBA reaction also includes a second NASBA primer,generally comprising DNA (although as for all the probes herein,including primers, nucleic acid analogs may also be used). This secondNASBA primer has a sequence at its 3′ end that is substantiallycomplementary to the Tend of the second template, and also contains anantisense sequence for a functional promoter and the antisense sequenceof a transcription initiation site. Thus, this primer sequence, whenused as a template for synthesis of the third DNA template, containssufficient information to allow specific and efficient binding of an RNApolymerase and initiation of transcription at the desired site.Preferred embodiments utilizes the antisense promoter and transcriptioninitiation site are that of the T7 RNA polymerase, although other RNApolymerase promoters and initiation sites can be used as well, asoutlined below.

The second primer hybridizes to the second template, and a DNApolymerase, also termed a “DNA-directed DNA polymerase”, also present inthe reaction, synthesizes a third template (a second newly synthesizedDNA strand), resulting in second hybridization complex comprising twonewly synthesized DNA strands.

Finally, the inclusion of an RNA polymerase and the required fourribonucleoside triphosphates (ribonucleotides or NTPs) results in thesynthesis of an RNA strand (a third newly synthesized strand that isessentially the same as the first template). The RNA polymerase,sometimes referred to herein as a “DNA-directed RNA polymerase”,recognizes the promoter and specifically initiates RNA synthesis at theinitiation site. In addition, the RNA polymerase preferably synthesizesseveral copies of RNA per DNA duplex. Preferred RNA polymerases include,but are not limited to, T7 RNA polymerase, and other bacteriophage RNApolymerases including those of phage T3, phage φII, Salmonella phagesp6, or Pseudomonase phage gh-1.

Accordingly, the NASBA reaction requires, in no particular order, afirst NASBA primer, a second NASBA primer comprising an antisensesequence of an RNA polymerase promoter, an RNA polymerase thatrecognizes the promoter, a reverse transcriptase, a DNA polymerase, anRNA degrading enzyme, NTPs and dNTPs, in addition to the detectioncomponents outlined below.

These components result in a single starting RNA template generating asingle DNA duplex; however, since this DNA duplex results in thecreation of multiple RNA strands, which can then be used to initiate thereaction again, amplification proceeds rapidly.

As outlined herein, the detection of the newly synthesized strands canproceed in several ways. Direct detection can be done in the detectionmodule when the newly synthesized strands comprise ETM labels, either byincorporation into the primers or by incorporation of modified labellednucleotides into the growing strand. Alternatively, as is more fullyoutlined below, indirect detection of unlabelled strands (which nowserve as “targets” In the detection mode) can occur using a variety ofsandwich assay configurations. As will be appreciated by those in theart, it is preferable to detect DNA strands during NASBA since thepresence of the ribonuclease makes the RNA strands potentially labile.

In a preferred embodiment, the amplification technique is signalamplification. Signal amplification involves the use of limited numberof target molecules as templates to either generate multiple signallingprobes or allow the use of multiple signalling probes. Signalamplification strategies include LCR, CPT, and the use of amplificationprobes in sandwich assays.

In a preferred embodiment, the signal amplification technique is LCR.The method can be run in two different ways; in a first embodiment, onlyone strand of a target sequence is used as a template for ligation;alternatively, both strands may be used. See generally U.S. Pat. Nos.5,185,243 and 5,573,907; EP 0 320 308 B1; EP 0 336 731 B1; EP 0 439 182B1; WO 90/01069; WO 89/12696; and WO 89/09835, and U.S. Ser. Nos.60/078,102 and 60/073,011, all of which are incorporated by reference.

In a preferred embodiment, the single-stranded target sequence comprisesa first target domain and a second target domain, and a first LCR primerand a second LCR primer nucleic acids are added, that are substantiallycomplementary to its respective target domain and thus will hybridize tothe target domains. These target domains may be directly adjacent, i.e.contiguous, or separated by a number of nucleotides. If they arenon-contiguous, nucleotides are added along with means to joinnucleotides, such as a polymerase, that will add the nucleotides to oneof the primers. The two LCR primers are then covalently attached, forexample using a ligase enzyme such as is known in the art. This forms afirst hybridization complex comprising the ligated probe and the targetsequence. This hybridization complex is then denatured (disassociated),and the process is repeated to generate a pool of ligated probes. Inaddition, it may be desirable to have the detection probes, describedbelow, comprise a mismatch at the probe junction site, such that thedetection probe cannot be used as a template for ligation.

In a preferred embodiment, LCR is done for two strands of adouble-stranded target sequence. The target sequence is denatured, andtwo sets of probes are added: one set as outlined above for one strandof the target, and a separate set (i.e. third and fourth primer robenucleic acids) for the other strand of the target. In a preferredembodiment, the first and third probes will hybridize, and the secondand fourth probes will hybridize, such that amplification can occur.That is, when the first and second probes have been attached, theligated probe can now be used as a template, in addition to the secondtarget sequence, for the attachment of the third and fourth probes.Similarly, the ligated third and fourth probes will serve as a templatefor the attachment of the first and second probes, in addition to thefirst target strand. In this way, an exponential, rather than just alinear, amplification can occur.

Again, as outlined above, the detection of the LCR reaction can occurdirectly, in the case where one or both of the primers comprises atleast one ETM, or indirectly, using sandwich assays, through the use ofadditional probes; that is, the ligated probes can serve as targetsequences, and detection may utilize amplification probes, captureprobes, capture extender probes, label probes, and label extenderprobes, etc.

In a preferred embodiment, the signal amplification technique is CPT.CPT technology is described in a number of patents and patentapplications, including U.S. Pat. Nos. 5,011,769, 5,403,711, 5,660,988,and 4,876,187, and PCT published applications WO 95/05480, WO 95/1416,and WO 95/00667, and U.S. Ser. No. 09/014,304, all of which areexpressly incorporated by reference in their entirety.

Generally, CPT may be described as follows. A CPT primer (also sometimesreferred to herein as a “scissile primer”), comprises two probesequences separated by a scissile linkage. The CPT primer issubstantially complementary to the target sequence and thus willhybridize to it to form a hybridization complex. The scissile linkage iscleaved, without cleaving the target sequence, resulting in the twoprobe sequences being separated. The two probe sequences can thus bemore easily disassociated from the target, and the reaction can berepeated any number of times. The cleaved primer is then detected asoutlined herein.

By “scissile linkage” herein is meant a linkage within the scissileprobe that can be cleaved when the probe is part of a hybridizationcomplex, that is, when a double-stranded complex is formed. It isimportant that the scissile linkage cleave only the scissile probe andnot the sequence to which it is hybridized (i.e. either the targetsequence or a probe sequence), such that the target sequence may bereused in the reaction for amplification of the signal. As used herein,the scissile linkage, is any connecting chemical structure which joinstwo probe sequences and which is capable of being selectively cleavedwithout cleavage of either the probe sequences or the sequence to whichthe scissile probe is hybridized. The scissile linkage may be a singlebond, or a multiple unit sequence. As will be appreciated by those inthe art, a number of possible scissile linkages may be used.

In a preferred embodiment, the scissile linkage comprises RNA. Thissystem, previously described in as outlined above, is based on the factthat certain double-stranded nucleases, particularly ribonucleases, willnick or excise RNA nucleosides from a RNA:DNA hybridization complex. Ofparticular use in this embodiment is RNAseH, Exo III, and reversetranscriptase.

In one embodiment, the entire scissile probe is made of RNA, the nickingis facilitated especially when carried out with a double-strandedribonuclease, such as RNAseH or Exo III. RNA probes made entirely of RNAsequences are particularly useful because first, they can be more easilyproduced enzymatically, and second, they have more cleavage sites whichare accessible to nicking or cleaving by a nicking agent, such as theribonucleases. Thus, scissile probes made entirely of RNA do not rely ona scissile linkage since the scissile linkage is inherent in the probe.

In a preferred embodiment, when the scissile linkage is a nucleic acidsuch as RNA, the methods of the invention may be used to detectmismatches, as is generally described in U.S. Pat. No. 5,660,988, and WO95/14106, hereby expressly incorporated by reference. These mismatchdetection methods are based on the fact that RNAseH may not bind toand/or cleave an RNA:DNA duplex if there are mismatches present in thesequence. Thus, in the NA₁-R-NA₂ embodiments, NA₁, and NA₂ are non-RNAnucleic acids, preferably DNA. Preferably, the mismatch is within theRNA:DNA duplex, but in some embodiments the mismatch is present in anadjacent sequence very close to the desired sequence, close enough toaffect the RNAseH (generally within one or two bases). Thus, in thisembodiment, the nucleic acid scissile linkage is designed such that thesequence of the scissile linkage reflects the particular sequence to bedetected, i.e. the area of the putative mismatch.

In some embodiments of mismatch detection, the rate of generation of thereleased fragments is such that the methods provide, essentially, ayes/no result, whereby the detection of the virtually any releasedfragment indicates the presence of the desired target sequence.Typically, however, when there is only a minimal mismatch (for example,a 1-, 2- or 3-base mismatch, or a 3-base defection), there is somegeneration of cleaved sequences even though the target sequence is notpresent. Thus, the rate of generation of cleaved fragments, and/or thefinal amount of cleaved fragments, is quantified to indicate thepresence or absence of the target. In addition, the use of secondary andtertiary scissile probes may be particularly useful in this embodiment,as this can amplify the differences between a perfect match and amismatch. These methods may be particularly useful in the determinationof homozygotic or heterozygotic states of a patient.

In this embodiment, it is an important feature of the scissile linkagethat its length is determined by the suspected difference between thetarget and the probe. In particular, this means that the scissilelinkage must be of sufficient length to encompass the suspecteddifference, yet short enough the scissile linkage cannot inappropriately“specifically hybridize” to the selected nucleic acid molecule when thesuspected difference is present; such inappropriate hybridization wouldpermit excision and thus cleavage of scissile linkages even though theselected nucleic acid molecule was not fully complementary to thenucleic acid probe. Thus in a preferred embodiment, the scissile linkageis between 3 to 5 nucleotides in length, such that a suspectednucleotide difference from 1 nucleotide to 3 nucleotides is encompassedby the scissile linkage, and 0, 1 or 2 nucleotides are on either side ofthe difference.

Thus, when the scissile linkage is nucleic acid, preferred embodimentsutilize from 1 to about 100 nucleotides, with from about 2 to about 20being preferred and from about 5 to about 10 being particularlypreferred.

CPT may be done enzymatically or chemically. That is, in addition toRNAseH, there are several other cleaving agents which may be useful incleaving RNA (or other nucleic acid) scissile bonds. For example,several chemical nucleases have been reported; see for example Sigman etal., Annu. Rev. Biochem. 1990, 59, 207-236; Sigman et al., Chem. Rev.1993, 93, 2295-2316; Bashkin et al., J. Org. Chem. 1990, 55, 5125-5132;and Sigman et al., Nucleic Acids and Molecular Biology, vol. 3, F.Eckstein and D. M. J. Lilley (Eds), Springer-Verlag, Heidelberg 1989,pp. 13-27; all of which are hereby expressly incorporated by reference.

Specific RNA hydrolysis is also an active area; see for example Chin,Acc. Chem. Res. 1991, 24, 145152; Breslow et al., Tetrahedron, 1991, 47,2365-2376; Anslyn et al., Angew. Chem. Int. Ed. Engl., 1997, 36,432-450; and references therein, all of which are expressly incorporatedby reference. Reactive phosphate centers are also of interest indeveloping scissile linkages, see Hendry et al., Prog. Inorg. Chem.:Bioinorganic Chem. 1990, 31, 201-258 also expressly incorporated byreference.

Current approaches to site-directed RNA hydrolysis include theconjugation of a reactive moiety capable of cleaving phosphodiesterbonds to a recognition element capable of sequence-specificallyhybridizing to RNA. In most cases, a metal complex is covalentlyattached to a DNA strand which forms a stable heteroduplex. Uponhybridization, a Lewis acid is placed in close proximity to the RNAbackbone to effect hydrolysis; see Magda et al., J. Am. Chem. Soc. 1994,116, 7439; Hall et al., Chem. Biology 1994, 1, 185-190; Bashkin et al.,J. Am. Chem. Soc. 1994, 116, 5981-5982; Hall et al., Nucleic Acids Res.1996, 24, 3522; Magda et al., J. Am. Chem. Soc. 1997, 119, 2293; andMagda et al., J. Am. Chem. Soc. 1997, 119, 6947, all of which areexpressly incorporated by reference.

In a similar fashion, DNA-polyamine conjugates have been demonstrated toinduce site-directed RNA strand scission; see for example, Yoshinari etal., J. Am. Chem. Soc. 1991, 113, 5899-5901; Endo et al., J. Org. Chem.1997, 62, 846; and Barbier et al., J. Am. Chem. Soc. 1992, 114,3511-3515, all of which are expressly incorporated by reference.

In a preferred embodiment, the scissile linkage is not necessarily RNA.For example, chemical cleavage moieties may be used to cleave basicsites in nucleic acids; see Belmont, et al., New J. Chem. 1997, 21,47-54; and references therein, all of which are expressly incorporatedherein by reference. Similarly, photocleavable moieties, for example,using transition metals, may be used; see Moucheron, et al., Inorg.Chem. 1997, 36, 584-592, hereby expressly by reference.

Other approaches rely on chemical moieties or enzymes; see for exampleKeck et al., Biochemistry 1995, 34, 12029-12037; Kirk et al., Chem.Commun. 1998, in press; cleavage of G-U basepairs by metal complexes;see Biochemistry, 1992, 31, 5423-5429; diamine complexes for cleavage ofRNA; Komiyama, et al., J. Org. Chem. 1997, 62, 2155-2160; and Chow etal., Chem. Rev. 1997, 97, 14891513, and references therein, all of whichare expressly incorporated herein by reference.

The first step of the CPT method requires hybridizing a primary scissileprimer (also called a primary scissile probe) obe to the target. This ispreferably done at a temperature that allows both the binding of thelonger primary probe and disassociation of the shorter cleaved portionsof the primary probe, as will be appreciated by those in the art. Asoutlined herein, this may be done in solution, or either the target orone or more of the scissile probes may be attached to a solid support.For example, it is possible to utilize “anchor probes” on a solidsupport or the electrode which are substantially complementary to aportion of the target sequence, preferably a sequence that is not thesame sequence to which a scissile probe will bind.

Similarly, as outlined herein, a preferred embodiment has one or more ofthe scissile probes attached to a solid support such as a bead. In thisembodiment, the soluble target diffuses to allow the formation of thehybridization complex between the soluble target sequence and thesupport-bound scissile probe. In this embodiment, it may be desirable toinclude additional scissile linkages in the scissile probes to allow therelease of two or more probe sequences, such that more than one probesequence per scissile probe may be detected, as is outlined below, inthe interests of maximizing the signal.

In this embodiment (and in other techniques herein), preferred methodsutilize cutting or shearing techniques to cut the nucleic acid samplecontaining the target sequence into a size that will allow sufficientdiffusion of the target sequence to the surface of a bead. This may beaccomplished by shearing the nucleic acid through mechanical forces orby cleaving the nucleic acid using restriction endonucleases.Alternatively, a fragment containing the target may be generated usingpolymerase, primers and the sample as a template, as in polymerase chainreaction (PCR). In addition, amplification of the target using PCR orLCR or related methods may also be done; this may be particularly usefulwhen the target sequence is present in the sample at extremely low copynumbers.

Similarly, numerous techniques are known in the art to increase the rateof mixing and hybridization including agitation, heating, techniquesthat increase the overall concentration such as precipitation, drying,dialysis, centrifugation, electrophoresis, magnetic bead concentration,etc.

In general, the scissile probes are introduced in a molar excess totheir targets (including both the target sequence or other scissileprobes, for example when secondary or tertiary scissile probes areused), with ratios of scissile probe:target of at least about 100:1being preferred, at least about 1000:1 being particularly preferred, andat least about 10,000:1 being especially preferred. In some embodimentsthe excess of probe:target will be much greater. In addition, ratiossuch as these may be used for all the amplification techniques outlinedherein.

Once the hybridization complex between the primary scissile probe andthe target has been formed, the complex is subjected to cleavageconditions. As will be appreciated, this depends on the composition ofthe scissile probe; if it is RNA, RNAseH is introduced. It should benoted that under certain circumstances, such as is generally outlined inWO 95/00666 and WO 95/00667, hereby incorporated by reference, the useof a double-stranded binding agent such as RNAseH may allow the reactionto proceed even at temperatures above the Tm of the primary probe:targethybridization complex. Accordingly, the addition of scissile probe tothe target can be done either first, and then the cleavage agent orcleavage conditions introduced, or the probes may be added in thepresence of the cleavage agent or conditions.

The cleavage conditions result in the separation of the two (or more)probe sequences of the primary scissile probe. As a result, the shorterprobe sequences will no longer remain hybridized to the target sequence,and thus the hybridization complex will disassociate, leaving the targetsequence intact. The optimal temperature for carrying out the CPTreactions is generally from about 5° C. to about 25° C. below themelting temperatures of the probe:target hybridization complex. Thisprovides for a rapid rate of hybridization and high degree ofspecificity for the target sequence. The Tm of any particularhybridization complex depends on salt concentration, G-C content, andlength of the complex, as is known in the art.

During the reaction, as for the other amplification techniques herein,it may be necessary to suppress cleavage of the probe, as well as thetarget sequence, by nonspecific nucleases. Such nucleases are generallyremoved from the sample during the isolation of the DNA by heating orextraction procedures. A number of inhibitors of single-strandednucleases such as vanadate, inhibitors it-ACE and RNAsin, a placentalprotein, do not affect the activity of RNAseH. This may not be necessarydepending on the purity of the RNAseH and/or the target sample.

These steps are repeated by allowing the reaction to proceed for aperiod of time. The reaction is usually carried out for about 15 minutesto about 1 hour. Generally, each molecule of the target sequence willturnover between 100 and 1000 times in this period, depending on thelength and sequence of the probe, the specific reaction conditions, andthe cleavage method. For example, for each copy of the target sequencepresent in the test sample 100 to 1000 molecules will be cleaved byRNAseH. Higher levels of amplification can be obtained by allowing thereaction to proceed longer, or using secondary, tertiary, or quaternaryprobes, as is outlined herein.

Upon completion of the reaction, generally determined by time or amountof cleavage, the uncleaved scissile probes must be removed orneutralized prior to detection, such that the uncleaved probe does notbind to a detection probe, causing false positive signals. This may bedone in a variety of ways, as is generally described below.

In a preferred embodiment, the separation is facilitated by the use of asolid support (either an internal surface of the device or beads trappedin the device) containing the primary probe. Thus, when the scissileprobes are attached to the solid support, the flow of the sample pastthis solid support can result in the removal of the uncleaved probes.

In a preferred embodiment, the separation is based on gelelectrophoresis of the reaction products to separate the longeruncleaved probe from the shorter cleaved probe sequences as is known inthe art and described herein.

In a preferred embodiment, the separation is based on strong acidprecipitation. This is useful to separate long (generally greater than50 nucleotides) from smaller fragments (generally about 10 nucleotides).The introduction of a strong acid such as trichloroacetic acid into thesolution (generally from a storage module) causes the longer probe toprecipitate, while the smaller cleaved fragments remain in solution. Theuse of fits or filters can to remove the precipitate, and the cleavedprobe sequences can be quantitated.

In a preferred embodiment, the scissile probe contains both an ETM andan affinity binding ligand or moiety, such that an affinity support isused to carry out the separation. In this embodiment, it is importantthat the ETM used for detection is not on the same probe sequence thatcontains the affinity moiety, such that removal of the uncleaved probe,and the cleaved probe containing the affinity moiety, does not removeall the detectable ETMs. Alternatively, the scissile probe may notcontain a covalently attached ETM, but just an affinity label. Suitableaffinity moieties include, but are not limited to, biotin, avidin,streptavidin, lectins, haptens, antibodies, etc. The binding partner ofthe affinity moiety is attached to a solid support (again, either aninternal surface of the device or to beads trapped within the device)and the flow of the sample past this support is used to pull out theuncleaved probes, as is known in the art. The cleaved probe sequences,which do not contain the affinity moiety, remain in solution and thencan be detected as outlined below.

In a preferred embodiment, similar to the above embodiment, a separationsequence of nucleic acid is included in the scissile probe, which is notcleaved during the reaction. A nucleic acid complementary to theseparation sequence is attached to a solid support and serves as acatcher sequence. Preferably, the separation sequence is added to thescissile probes, and is not recognized by the target sequence, such thata generalized catcher sequence may be utilized in a variety of assays.

In a preferred embodiment, the uncleaved probe is neutralized by theaddition of a substantially complementary neutralization nucleic acid,generally from a storage module. This is particularly useful inembodiments utilizing capture sequences, separation sequences, andone-step systems, as the complement to a probe containing capturesequences forms hybridization complexes that are more stable due to itslength than the cleaved probe sequence:detection probe complex. As willbe appreciated by those in the art, complete removal of the uncleavedprobe is not required, since detection is based on electron transferthrough nucleic acid; rather, what is important is that the uncleavedprobe is not available for binding to a detection electrode probespecific for cleaved sequences. Thus, in one embodiment, this stepoccurs in the detection module and the neutralization nucleic acid is adetection probe on the surface of the electrode, at a separate“address”, such that the signal from the neutralization hybridizationcomplex does not contribute to the signal of the cleaved fragments.Alternatively, the neutralization nucleic acid may be attached to asolid support; the sample flowed past the neutralization surface toquench the reaction, and thus do not enter the detection module.

After removal or neutralization of the uncleaved probe, detectionproceeds via the addition of the cleaved probe sequences to thedetection module, as outlined below, which can utilize either“mechanism-1” or “mechanism-2” systems.

In a preferred embodiment, no higher order probes are used, anddetection is based on the probe sequence(s) of the primary primer. In apreferred embodiment, at least one, and preferably more, secondaryprobes (also referred to herein as secondary primers) are used. Thesecondary scissile probes may be added to the reaction in several ways.It is important that the secondary scissile probes be prevented fromhybridizing to the uncleaved primary probes, as this results in thegeneration of false positive signal. In a preferred embodiment, theprimary and secondary probes are bound to solid supports. It is onlyupon hybridization of the primary probes with the target, resulting incleavage and release of primary probe sequences from the bead, that thenow diffusible primary probe sequences may bind to the secondary probes.In turn, the primary probe sequences serve as targets for the secondaryscissile probes, resulting in cleavage and release of secondary probesequences.

In an alternate embodiment, the complete reaction is done in solution.In this embodiment, the primary probes are added, the reaction isallowed to proceed for some period of time, and the uncleaved primaryscissile probes are removed, as outlined above. The secondary probes arethen added, and the reaction proceeds. The secondary uncleaved probesare then removed, and the cleaved sequences are detected as is generallyoutlined herein. In a preferred embodiment, at least one, and preferablymore, tertiary probes are used. The tertiary scissile probes may beadded to the reaction in several ways. It is important that the tertiaryscissile probes be prevented from hybridizing to the uncleaved secondaryprobes, as this results in the generation of false positive signal.These methods are generally done as outlined above. Similarly,quaternary probes can be used as above.

Thus, CPT requires, again in no particular order, a first CPT primercomprising a first probe sequence, a scissile linkage and a second probesequence; and a cleavage agent.

In this manner, CPT results in the generation of a large amount ofcleaved primers, which then can be detected as outlined below.

In a preferred embodiment, the signal amplification technique is a“sandwich” assay, as is generally described in U.S. Ser. No. 60/073,011and in U.S. Pat. Nos. 5,681,702, 5,597,909, 5,545,730, 5,594,117,5,591,584, 5,571,670, 5,580,731, 5,571,670, 5,591,584, 5,624,802,5,635,352, 5,594,118, 5,359,100, 5,124,246 and 5,681,697, all of whichare hereby incorporated by reference. Although sandwich assays do notresult in the alteration of primers, sandwich assays can be consideredsignal amplification techniques since multiple signals (i.e. labelprobes) are bound to a single target, resulting in the amplification ofthe signal. Sandwich assays are used when the target sequence compriseslittle or no ETM labels; that is, when a secondary probe, comprising theETM labels, is used to generate the signal.

As discussed herein, it should be noted that the sandwich assays can beused for the detection of primary target sequences (e.g. from a patientsample), or as a method to detect the product of an amplificationreaction as outlined above; thus for example, any of the newlysynthesized stir ands outlined above, for example using PCR, LCR, NASBA,SDA, etc., may be used as the “target sequence” in a sandwich assay.

Generally, sandwich signal amplification techniques may be described asfollows. The reactions described below can occur either in the reactionmodule, with subsequent transfer to the detection module for detection,or in the detection module with the addition of the required components;for clarity, these are discussed together.

As a preliminary matter, as is more fully described below, captureextender probes may be added to the target sequence for attachment to anelectrode in the detection module.

The methods include the addition of an amplifier probe, which ishybridized to the target sequence, either directly, or through the useof one or more label extender probes, which serves to allow “generic”amplifier probes to be made. Preferably, the amplifier probe contains amultiplicity of amplification sequences, although in some embodiments,as described below, the amplifier probe may contain only a singleamplification sequence, or at least two amplification sequences. Theamplifier probe may take on a number of different forms; either abranched conformation, a dendrimer conformation, or a linear “string” ofamplification sequences. Label probes comprising ETMs then hybridize tothe amplification sequences (or in some cases the label probes hybridizedirectly to the target sequence), and the ETMs are detected using theelectrode, as is more fully outlined below.

As will be appreciated by those in the art, the systems of the inventionmay take on a large number of different configurations. In general,there are three types of systems that can be used: (1) “non-sandwich”systems (also referred to herein as “direct” detection) in which thetarget sequence itself is labeled with ETMs (again, either because theprimers comprise ETMs or due to the incorporation of ETMs into the newlysynthesized strand); (2) systems in which label probes directly bind tothe target analytes; and (3) systems in which label probes areindirectly bound to the target sequences, for example through the use ofamplifier probes.

Accordingly, the present invention provides compositions comprising anamplifier probe. By “amplifier probe” or “nucleic acid multimer” or“amplification multimer” or grammatical equivalents herein is meant anucleic acid probe that is used to facilitate signal amplification.Amplifier probes comprise at least a first single-stranded nucleic acidprobe sequence, as defined below, and at least one singlestrandednucleic acid amplification sequence, with a multiplicity ofamplification sequences being preferred.

Amplifier probes comprise a first probe sequence that is used, eitherdirectly or indirectly, to hybridize to the target sequence. That is,the amplifier probe itself may have a first probe sequence that issubstantially complementary to the target sequence, or it has a firstprobe sequence that is substantially complementary to a portion of anadditional probe, in this case called a label extender probe, that has afirst portion that is substantially complementary to the targetsequence. In a preferred embodiment, the first probe sequence of theamplifier probe is substantially complementary to the target sequence.

In general, as for all the probes herein, the first probe sequence is ofa length sufficient to give specificity and stability. Thus generally,the probe sequences of the invention that are designed to hybridize toanother nucleic acid (i.e. probe sequences, amplification sequences,portions or domains of larger probes) are at least about 5 nucleosideslong, with at least about 10 being preferred and at least about 15 beingespecially preferred.

In a preferred embodiment, the amplifier probes, or any of the otherprobes of the invention, may form hairpin stem-loop structures in theabsence of their target. The length of the stem double-stranded sequencewill be selected such that the hairpin structure is not favored in thepresence of target. The use of these type of probes, in the systems ofthe invention or in any nucleic acid detection systems, can result in asignificant decrease in non-specific binding and thus an increase in thesignal to noise ratio.

Generally, these hairpin structures comprise four components. The firstcomponent is a target binding sequence, i.e. a region complementary tothe target (which may be the sample target sequence or another probesequence to which binding is desired), that is about 10 nucleosideslong, with about 15 being preferred. The second component is a loopsequence, that can facilitate the formation of nucleic acid loops.Particularly preferred in this regard are repeats of GTC, which has beenidentified in Fragile X Syndrome as forming turns. (When PNA analogs areused, turns comprising proline residues may be preferred). Generally,from three to five repeats are used, with four to five being preferred.The third component is a self-complementary region, which has a firstportion that is complementary to a portion of the target sequence regionand a second portion that comprises a first portion of the label probebinding sequence. The fourth component is substantially complementary toa label probe (or other probe, as the case may be). The fourth componentfurther comprises a “sticky end”, that is, a portion that does nothybridize to any other portion of the probe, and preferably containsmost, if not all, of the ETMs. As will be appreciated by those in theart, any or all of the probes described herein may be configured to formhairpins in the absence of their targets, including the amplifier,capture, capture extender, label and label extender probes.

In a preferred embodiment, several different amplifier probes are used,each with first probe sequences that will hybridize to a differentportion of the target sequence. That is, there is more than one level ofamplification; the amplifier probe provides an amplification of signaldue to a multiplicity of labelling events, and several differentamplifier probes, each with this multiplicity of labels, for each targetsequence is used. Thus, preferred embodiments utilize at least twodifferent pools of amplifier probes, each pool having a different probesequence for hybridization to different portions of the target sequence;the only real limitation on the number of different amplifier probeswill be the length of the original target sequence. In addition, it isalso possible that the different amplifier probes contain differentamplification sequences, although this is generally not preferred.

In a preferred embodiment, the amplifier probe does not hybridize to thesample target sequence directly, but instead hybridizes to a firstportion of a label extender probe. This is particularly useful to allowthe use of “generic” amplifier probes, that is, amplifier probes thatcan be used with a variety of different targets. This may be desirablesince several of the amplifier probes require special synthesistechniques. Thus, the addition of a relatively short probe as a labelextender probe is preferred. Thus, the first probe sequence of theamplifier probe is substantially complementary to a first portion ordomain of a first label extender single-stranded nucleic acid probe. Thelabel extender probe also contains a second portion or domain that issubstantially complementary to a portion of the target sequence. Both ofthese portions are preferably at least about 10 to about 50 nucleotidesin length, with a range of about 15 to about 30 being preferred. Theterms “first” and “second” are not meant to confer an orientation of thesequences with respect to the 5′→3′ orientation of the target or probesequences. For example, assuming a 5′→3′ orientation of thecomplementary target sequence, the first portion may be located either5′ to the second portion, or 3′ to the second portion. For convenienceherein, the order of probe sequences are generally shown from left toright.

In a preferred embodiment, more than one label extender probe-amplifierprobe pair may be used. That is, a plurality of label extender probesmay be used, each with a portion that is substantially complementary toa different portion of the target sequence; this can serve as anotherlevel of amplification. Thus, a preferred embodiment utilizes pools ofat least two label extender probes, with the upper limit being set bythe length of the target sequence.

In a preferred embodiment, more than one label extender probe is usedwith a single amplifier probe to reduce non-specific binding, as isgenerally outlined in U.S. Pat. No. 5,681,697, incorporated by referenceherein. In this embodiment, a first portion of the first label extenderprobe hybridizes to a first portion of the target sequence, and thesecond portion of the first label extender probe hybridizes to a firstprobe sequence of the amplifier probe. A first portion of the secondlabel extender probe hybridizes to a second portion of the targetsequence, and the second portion of the second label extender probehybridizes to a second probe sequence of the amplifier probe. These formstructures sometimes referred to as “cruciform” structures orconfigurations, and are generally done to confer stability when largebranched or dendrimeric, amplifier probes are used.

In addition, as will be appreciated by those in the art, the labelextender probes may interact with a preamplifier probe, described below,rather than the amplifier probe directly.

Similarly, as outlined above, a preferred embodiment utilizes severaldifferent amplifier probes, each with first probe sequences that willhybridize to a different portion of the label extender probe. Inaddition, as outlined above, it is also possible that the differentamplifier probes contain different amplification sequences, althoughthis is generally not preferred.

In addition to the first probe sequence, the amplifier probe alsocomprises at least one amplification sequence. An “amplificationsequence” or “amplification segment” or grammatical equivalents hereinis meant a sequence that is used, either directly or indirectly, to bindto a first portion of a label probe as is more fully described below(although in some cases the amplification sequence may bind to adetection probe). Preferably, the amplifier probe comprises amultiplicity of amplification sequences, with from about 3 to about 1000being preferred, from about 10 to about 100 being particularlypreferred, and about 50 being especially preferred. In some cases, forexample when linear amplifier probes are used, from 1 to about 20 ispreferred with from about 5 to about 10 being particularly preferred.

The amplification sequences may be linked to each other in a variety ofways, as will be appreciated by those in the art. They may be covalentlylinked directly to each other, or to intervening sequences or chemicalmoieties, through nucleic acid linkages such as phosphodiester bonds,PNA bonds, etc., or through interposed linking agents such amino acid,carbohydrate or polyol bridges, or through other cross-linking agents orbinding partners. The site(s) of linkage may be at the ends of asegment, and/or at one or more internal nucleotides in the strand. In apreferred embodiment, the amplification sequences are attached vianucleic acid linkages.

In a preferred embodiment, branched amplifier probes are used, as aregenerally described in U.S. Pat. No. 5,124,246, hereby incorporated byreference. Branched amplifier probes may take on “fork-like” or“comb-like” conformations. “Fork-like” branched amplifier probesgenerally have three or more oligonucleotide segments emanating from apoint of origin to form a branched structure. The point of origin may beanother nucleotide segment or a multifunctional molecule to which atleast three segments can be covalently or tightly bound. “Comb-like”branched amplifier probes have a linear backbone with a multiplicity ofsidechain oligonucleotides extending from the backbone. In eitherconformation, the pendant segments will normally depend from a modifiednucleotide or other organic moiety having the appropriate functionalgroups for attachment of oligonucleotides. Furthermore, in eitherconformation, a large number of amplification sequences are availablefor binding, either directly or indirectly, to detection probes. Ingeneral, these structures are made as is known in the art, usingmodified multifunctional nucleotides, as is described in U.S. Pat. Nos.5,635,352 and 5,124,246, among others.

In a preferred embodiment, dendrimer amplifier probes are used, as aregenerally described in U.S. Pat. No. 5,175,270, hereby expresslyincorporated by reference. Dendrimeric amplifier probes haveamplification sequences that are attached via hybridization, and thushave portions of double-stranded nucleic acid as a component of theirstructure. The outer surface of the dendrimer amplifier probe has amultiplicity of amplification sequences.

In a preferred embodiment, linear amplifier probes are used, that haveindividual amplification sequences linked end-to-end either directly orwith short intervening sequences to form a polymer. As with the otheramplifier configurations, there may be additional sequences or moietiesbetween the amplification sequences. In addition, as outlined herein,linear amplification probes may form hairpin stem-loop structures.

In one embodiment, the linear amplifier probe has a single amplificationsequence. This may be useful when cycles of hybridization/disassociationoccurs, forming a pool of amplifier probe that was hybridized to thetarget and then removed to allow more probes to bind, or when largenumbers of ETMs are used for each label probe. However, in a preferredembodiment, linear amplifier probes comprise a multiplicity ofamplification sequences.

In addition, the amplifier probe may be totally linear, totallybranched, totally dendrimeric, or any combination thereof.

The amplification sequences of the amplifier probe are used, eitherdirectly or indirectly, to bind to a label probe to allow detection. Ina preferred embodiment, the amplification sequences of the amplifierprobe are substantially complementary to a first portion of a labelprobe. Alternatively, amplifier extender probes are used, that have afirst portion that binds to the amplification sequence and a secondportion that binds to the first portion of the label probe.

In addition, the compositions of the invention may include“preamplifier” molecules, which serves a bridging moiety between thelabel extender molecules and the amplifier probes. In this way, moreamplifier and thus more ETMs are ultimately bound to the detectionprobes. Preamplifier molecules may be either linear or branched, andtypically contain in the range of about 30-3000 nucleotides.

Thus, label probes are either substantially complementary to anamplification sequence or to a portion of the target sequence.Accordingly, the label probes can be configured in a variety of ways, asis generally described herein, depending on whether a “mechanism-1” or“mechanism-2” detection system is utilized, as described below.

Detection of the amplification reactions of the invention, including thedirect detection of amplification products and indirect detectionutilizing label probes (i.e. sandwich assays), is done by detectingassay complexes comprising ETMs, which can be attached to the assaycomplex in a variety of ways, as is more fully described below.

In addition, as described in U.S. Pat. No. 5,587,128, the reactionchamber may comprise a composition, either in solution or adhered to thesurface of the reaction chamber, that prevents the inhibition of anamplification reaction by the composition of the well. For example, thewall surfaces may be coated with a silane, for example using asilanization reagent such as dimethylchlorosilane, or coated with asiliconizing reagent such as Aquasil™ or Surfacil™ (Pierce, Rockford,Ill.), which are organosilanes containing a hydrolyzable group. Thishydrolyzable group can hydrolyze in solution to form a silanol that canpolymerize and form a tightly bonded film over the surface of thechamber. The coating may also include a blocking agent that can reactwith the film to further reduce inhibition; suitable blocking agentsinclude amino acid polymers and polymers such as polyvinylpyrrolidone,polyadenylic acid and polymaleimide. Alternatively, for siliconsubstrates, a silicon oxide film may be provided on the walls, or thereaction chamber can be coated with a relatively inert polymer such as apolyvinylchloride. In addition, it may be desirable to add blockingpolynucleotides to occupy any binding sites on the surface of thechamber.

In this and other embodiments, a thermal module may be used, that iseither part of the reaction chamber or separate but can be brought intospatial proximity to the reaction module. The thermal module can includeboth heating and/or cooling capability. Suitable thermal modules aredescribed in U.S. Pat. Nos. 5,498,392 and 5,587,128, and WO 97/16561,incorporated by reference, and may comprise electrical resistanceheaters, pulsed lasers or other sources of electromagnetic energydirected to the reaction chamber. It should also be noted that whenheating elements are used, it may be desirable to have the reactionchamber be relatively shallow, to facilitate heat transfer; see U.S.Pat. No. 5,587,128.

In a preferred embodiment, the biological reaction chamber allowsenzymatic cleavage or alteration of the target analyte. For example,restriction endonucleases may be used to cleave target nucleic acidscomprising target sequences, for example genomic DNA, into smallerfragments to facilitate either amplification or detection.Alternatively, when the target analyte is a protein, it may be cleavedby a protease. Other types of enzymatic hydrolysis may also be done,depending on the composition of the target analyte. In addition, asoutlined herein, the target analyte may comprise an enzyme and thereaction chamber comprises a substrate that is then cleaved to form adetectable product.

In addition, in one embodiment the reaction module includes a chamberfor the physical alteration of all or part of the sample, for examplefor shearing genomic or large nucleic acids, nuclear lysis, ultrasound,etc.

In a preferred embodiment, the devices of the invention include at leastone fluid pump. Pumps generally fall into two categories: “on chip” and“off chip”; that is, the pumps (generally electrode based pumps) can becontained within the device itself, or they can be contained on anapparatus into which the device fits, such that alignment occurs of therequired flow channels to allow pumping of fluids.

In a preferred embodiment, the pumps are contained on the device itself.These pumps are generally electrode based pumps; that is, theapplication of electric fields can be used to move both chargedparticles and bulk solvent, depending on the composition of the sampleand of the device. Suitable on chip pumps include, but are not limitedto, electroosmotic (EO) pumps and electrohydrodynamic (EHD) pumps; theseelectrode based pumps have sometimes been referred to in the art as“electrokinetic (EK) pumps”. All of these pumps rely on configurationsof electrodes placed along a flow channel to result in the pumping ofthe fluids comprising the sample components. As is described in the art,the configurations for each of these electrode based pumps are slightlydifferent; for example, the effectiveness of an EHD pump depends on thespacing between the two electrodes, with the closer together they are,the smaller the voltage required to be applied to effect fluid flow.Alternatively, for EO pumps, the sampcing between the electrodes shouldbe larger, with up to one-half the length of the channel in which fluidsare being moved, since the electrode are only involved in applyingforce, and not, as in EHD, in creating charges on which the force willact.

In a preferred embodiment, an electroosmotic pump is used.Electroosmosis (EO) is based on the fact that the surface of manysolids, including quartz, glass and others, become variously charged,negatively or positively, in the presence of ionic materials. Thecharged surfaces will attract oppositely charged counterions in aqueoussolutions. Applying a voltage results in a migration of the counterionsto the oppositely chaged electrode, and moves the bulk of the fluid aswell. The volume flow rate is proportional to the current, and thevolume flow generated in the fluid is also proportional to the appliedvoltage. Electroosmostic flow is useful for liquids having someconductivity is and generally not applicable for non-polar solvents. EOpumps are described in U.S. Pat. Nos. 4,908,112 and 5,632,876, PCTUS95/14586 and WO97/43629, incorporated by reference.

In a preferred embodiment, an electrohydrodynamic (EHD) pump is used. InEHD, electrodes in contact with the fluid transfer charge when a voltageis applied. This charge transfer occurs either by transfer or removal ofan electron to or from the fluid, such that liquid flow occurs in thedirection from the charging electrode to the oppositely chargedelectrode. EHD pumps can be used to pump resistive fluids such asnon-polar solvents. EHD pumps are described in U.S. Pat. No. 5,632,876,hereby incorporated by reference.

The electrodes of the pumps preferably have a diameter from about 25microns to about 100 microns, more preferably from about 50 microns toabout 75 microns. Preferably, the electrodes protrude from the top of aflow channel to a depth of from about 5% to about 95% of the depth ofthe channel, with from about 25% to about 50% being preferred. Inaddition, as described in PCT US95/14586, an electrode-based internalpumping system can be integrated into the liquid distribution system ofthe devices of the invention with flow-rate control at multiple pumpsites and with fewer complex electronics if the pumps are operated byapplying pulsed voltages across the electrodes; this gives theadditional advantage of ease of integration into high density systems,reductions in the amount of electrolysis that occurs at electrodes,reductions in thermal convenction near the electrodes, and the abilityto use simpler drivers, and the ability to use both simple and complexpulse wave geometries.

The voltages required to be applied to the electrodes cause fluid flowdepends on the geometry of the electrodes and the properties of thefluids to be moved. The flow rate of the fluids is a function of theamplitude of the applied voltage between electrode, the electrodegeometery and the fluid properties, which can be easily determined foreach fluid. Test voltages used may be up to about 1500 volts, but anoperating voltage of about 40 to 300 volts is desirable. An analogdriver is generally used to vary the voltage applied to the pump from aDC power source. A transfer function for each fluid is determinedexperimentally as that applied voltage that produces the desired flow orfluid pressure to the fluid being moved in the channel. However, ananalog driver is generally required for each pump along the channel andis suitable an operational amplifier.

In a preferred embodiment, a micromechanical pump is used, either on- oroff chip, as is known in the art.

In a preferred embodiment, an “off-chip” pump is used. For example, thedevices of the invention may fit into an apparatus or appliance that hasa nesting site for holding the device, that can register the ports (i.e.sample inlet ports, fluid inlet ports, and waste outlet ports) andelectrode leads. The apparatus can including pumps that can apply thesample to the device; for example, can force cellcontaining samples intocell lysis modules containing protrusions, to cause cell lysis uponapplication of sufficient flow pressure. Such pumps are well known inthe art.

In a preferred embodiment, the devices of the invention include at leastone fluid valve that can control the flow of fluid into or out of amodule of the device, or divert the flow into one or more channels. Avariety of valves are known in the art. For example, in one embodiment,the valve may comprise a capillary barrier, as generally described inPCT US97/07880, incorporated by reference. In this embodiment, thechannel opens into a larger space designed to favor the formation of anenergy minimizing liquid surface such as a meniscus at the opening.Preferably, capillary barriers include a dam that raises the verticalheight of the channel immediated before the opening into a larger spacesuch a chamber. In addition, as described in U.S. Pat. No. 5,858,195,incorporated herein by reference, a type of “virtual valve” can be used.

In a preferred embodiment, the devices of the invention include sealingports, to allow the introduction of fluids, including samples, into anyof the modules of the invention, with subsequent closure of the port toavoid the loss of the sample.

In a preferred embodiment, the devices of the invention include at leastone storage modules for assay reagents. These are connected to othermodules of the system using flow channels and may comprise wells orchambers, or extended flow channels. They may contain any number ofreagents, buffers, enzymes, electronic mediators, salts, etc., includingfreeze dried reagents.

In a preferred embodiment, the devices of the invention include a mixingmodule; again, as for storage modules, these may be extended flowchannels (particularly useful for timed mixing), wells or chambers.Particularly in the case of extended flow channels, there may beprotrusions on the side of the channel to cause mixing.

In a preferred embodiment, the devices of the invention include adetection module. The present invention is directed to methods andcompositions useful in the detection of biological target analytespecies such as nucleic acids and proteins. In general, the detectionmodule is based on work outlined in U.S. Pat. No. 5,591,578; 5,824,473;5,770,369; 5,705,348 and 5,780,234; U.S. Ser. Nos. 09/096,593;08/911,589; 09/135,183; and 60/105,875; and PCT applications US97/20014and US98/12082; all of which are hereby incorporated by reference intheir entirety. The system is generally described as follows. A targetanalyte is introduced to the detection module, and is combined withother components to form an assay complex in a variety of ways, as ismore fully outlined below. The assay complexes comprise electrontransfer moieties (ETMs), which can be attached to the assay complex ina variety of ways, as is more fully described below. In general, thereare two basic detection mechanisms. In a preferred embodiment, detectionof an ETM is based on electron transfer through the stacked π-orbitalsof double stranded nucleic acid. This basic mechanism is described inU.S. Pat. Nos. 5,591,578, 5,770,369, and 5,705,348 and PCT US97/20014and is termed “mechanism-1” herein. Briefly, previous work has shownthat electron transfer can proceed rapidly through the stackedn-orbitals of double stranded nucleic acid, and significantly moreslowly through single-stranded nucleic acid. Accordingly, this can serveas the basis of an assay. Thus, by adding ETMs (either covalently to oneof the strands or non-covalently to the hybridization complex throughthe use of hybridization indicators, described below) to a nucleic acidthat is attached to a detection electrode via a conductive oligomer,electron transfer between the ETM and the electrode, through the nucleicacid and conductive oligomer, may be detected.

This may be done where the target analyte is a nucleic acid;alternatively, a non-nucleic acid target analyte is used, with anoptional capture binding ligand (to attach the target analyte to thedetection electrode) and a soluble binding ligand that carries a nucleicacid “tail”, that can then bind either directly or indirectly to adetection probe on the surface to effect detection.

Alternatively, the ETM can be detected, not necessarily via electrontransfer through nucleic acid, but rather can be directly detected usingconductive oligomers; that is, the electrons from the ETMs need nottravel through the stacked n orbitals in order to generate a signal.Instead, the presence of ETMs on the surface of a SAM, that comprisesconductive oligomers, can be directly detected. This basic idea istermed “mechanism-2” herein. Thus, upon binding of a target analyte, asoluble binding ligand comprising an ETM is brought to the surface, anddetection of the ETM can proceed, putatively through the conductiveoligomer to the electrode. Essentially, the role of the SAM comprisingthe conductive oligomers is to “raise” the electronic surface of theelectrode, while still providing the benefits of shielding the electrodefrom solution components and reducing the amount of non-specific bindingto the electrodes. Viewed differently, the role of the binding ligand isto provide specificity for a recruitment of ETMs to the surface, wherethey can be detected using conductive oligomers with electronicallyexposed termini.

Thus, in either embodiment, an assay complex is formed that contains anETM, which is then detected using the detection electrode.

The present system finds particular utility in array formats, i.e.wherein there is a matrix of addressable microscopic detectionelectrodes (herein generally referred to “pads”, “addresses” or“micro-locations”).

Accordingly, the present invention provides methods for detecting targetanalytes in sample solutions using an electrode. If required, the targetanalyte is prepared using known techniques, generally within the devicesoutlined above. For example, the sample may be treated to lyse thecells, using known lysis buffers, sonication, electroporation, etc.,with purification occurring as needed, as will be appreciated by thosein the art.

The detection modules of the invention comprise electrodes. By“electrode” herein is meant a composition, which, when connected to anelectronic device, is able to sense a current or charge and convert itto a signal. Alternatively an electrode can be defined as a compositionwhich can apply a potential to and/or pass electrons to or from speciesin the solution. Thus, an electrode is an ETM as described herein.Preferred electrodes are known in the art and include, but are notlimited to, certain metals and their oxides, including gold; platinum;palladium; silicon; aluminum; metal oxide electrodes including platinumoxide, titanium oxide, tin oxide, indium tin oxide, palladium oxide,silicon oxide, aluminum oxide, molybdenum oxide (Mo₂O₆), tungsten oxide(WO₃) and ruthenium oxides; and carbon (including glassy carbonelectrodes, graphite and carbon paste). Preferred electrodes includegold, silicon, platinum, carbon and metal oxide electrodes, with goldbeing particularly preferred.

The electrodes described herein are depicted as a flat surface, which isonly one of the possible conformations of the electrode and is forschematic purposes only. The conformation of the electrode will varywith the detection method used. For example, flat planar electrodes maybe preferred for optical detection methods, or when arrays of nucleicacids are made, thus requiring addressable locations for both synthesisand detection. Alternatively, for single probe analysis, the electrodemay be in the form of a tube, with the SAMs comprising conductiveoligomers and nucleic acids bound to the inner surface. Electrode coilsmay be preferred in some embodiments as well. This allows a maximum ofsurface area containing the nucleic acids to be exposed to a smallvolume of sample.

The detection electrode comprises a self-assembled monolayer (SAM)comprising conductive oligomers. By “monolayer” or “self-assembledmonolayer” or “SAM” herein is meant a relatively ordered assembly ofmolecules spontaneously chemisorbed on a surface, in which the moleculesare oriented approximately parallel to each other and roughlyperpendicular to the surface. Each of the molecules includes afunctional group that adheres to the surface, and a portion thatinteracts with neighboring molecules in the monolayer to form therelatively ordered array. A “mixed” monolayer comprises a heterogeneousmonolayer, that is, where at least two different molecules make up themonolayer. The SAM may comprise conductive oligomers alone, or a mixtureof conductive oligomers and insulators. As outlined herein, theefficiency of target analyte binding (for example, oligonucleotidehybridization) may increase when the analyte is at a distance from theelectrode. Similarly, nonspecific binding of biomolecules, including thetarget analytes, to an electrode is generally reduced when a monolayeris present. Thus, a monolayer facilitates the maintenance of the analyteaway from the electrode surface. In addition, a monolayer serves to keepcharged species away from the surface of the electrode. Thus, this layerhelps to prevent electrical contact between the electrodes and the ETMs,or between the electrode and charged species within the solvent. Suchcontact can result in a direct “short circuit” or an indirect shortcircuit via charged species which may be present in the sample.Accordingly, the monolayer is preferably tightly packed in a uniformlayer on the electrode surface, such that a minimum of “holes” exist.The monolayer thus serves as a physical barrier to block solventaccesibility to the electrode.

In a preferred embodiment, the monolayer comprises conductive oligomers.By “conductive oligomer” herein is meant a substantially conductingoligomer, preferably linear, some embodiments of which are referred toin the literature as “molecular wires”. By “substantially conducting”herein is meant that the oligomer is capable of transfering electrons at100 Hz. Generally, the conductive oligomer has substantially overlappingπ-orbitals, i.e. conjugated π-orbitals, as between the monomeric unitsof the conductive oligomer, although the conductive oligomer may alsocontain one or more sigma (σ) bonds. Additionally, a conductive oligomermay be defined functionally by its ability to inject or receiveelectrons into or from an associated ETM. Furthermore, the conductiveoligomer is more conductive than the insulators as defined herein.Additionally, the conductive oligomers of the invention are to bedistinguished from electroactive polymers, that themselves may donate oraccept electrons.

In a preferred embodiment, the conductive oligomers have a conductivity,S, of from between about 10⁻⁶ about 10⁴Ω⁻¹ cm⁻¹, with from about 10⁻⁵ toabout 10³Ω⁻¹ cm⁻¹ being preferred, with these S values being calculatedfor molecules ranging from about 20 Å to about 200 Å. As describedbelow, insulators have a conductivity S of about 10⁻⁷Ω⁻¹ cm⁻¹ or lower,with less than about 10⁻⁸Ω⁻¹ cm⁻¹ being preferred. See generally Gardneret al., Sensors and Actuators A 51 (1995) 57-66, incorporated herein byreference.

Desired characteristics of a conductive oligomer include highconductivity, sufficient solubility in organic solvents and/or water forsynthesis and use of the compositions of the invention, and preferablychemical resistance to reactions that occur i) during binding ligandsynthesis (i.e. nucleic acid synthesis, such that nucleosides containingthe conductive oligomers may be added to a nucleic acid synthesizerduring the synthesis of the compositions of the invention, ii) duringthe attachment of the conductive oligomer to an electrode, or iii)during binding assays. In addition, conductive oligomers that willpromote the formation of self-assembled monolayers are preferred.

The oligomers of the invention comprise at least two monomeric subunits,as described herein. As is described more fully below, oligomers includehomo- and hetero-oligomers, and include polymers.

In a preferred embodiment, the conductive oligomer has the structuredepicted in Structure 1:

As will be understood by those in the art, all of the structuresdepicted herein may have additional atoms or structures; i.e. theconductive oligomer of Structure 1 may be attached to ETMs, such aselectrodes, transition metal complexes, organic ETMs, and metallocenes,and to binding ligands such as nucleic acids, or to several of these.Unless otherwise noted, the conductive oligomers depicted herein will beattached at the left side to an electrode; that is, as depicted inStructure 1, the left “Y” is connected to the electrode as describedherein. If the conductive oligomer is to be attached to a bindingligand, the right “Y”, if present, is attached to the binding ligandsuch as a nucleic acid, either directly or through the use of a linker,as is described herein.

In this embodiment, Y is an aromatic group, n is an integer front 1 to50, g is either 1 or zero, e is an integer from zero to 10, and m iszero or 1. When g is 1, B-D is a bond able to conjugate with neighboringbonds (herein referred to as a “conjugated bond”), preferably selectedfrom acetylene, alkene, substituted alkene, amide, azo, —C═N— (including—N═C—, —CR═N— and —N═CR—), —Si═Si—, and —Si═C— (including —C═Si—,—Si═CR— and —CR═Si—). When g is zero, e is preferably 1, D is preferablycarbonyl, or a heteroatom moiety, wherein the heteroatom is selectedfrom oxygen, sulfur, nitrogen, silicon or phosphorus. Thus, suitableheteroatom moieties include, but are not limited to, —NH and —NR,wherein R is as defined herein; substituted sulfur; sulfonyl (—SO₂—)sulfoxide (—SO—); phosphine oxide (—PO— and —RPO—); and thiophosphine(—PS— and —RPS—). However, when the conductive oligomer is to beattached to a gold electrode, as outlined below, sulfur derivatives arenot preferred.

By “aromatic group” or grammatical equivalents herein is meant anaromatic monocyclic or polycyclic hydrocarbon moiety generallycontaining 5 to 14 carbon atoms (although larger polycyclic ringsstructures may be made) and any carbocylic ketone or thioketonederivative thereof, wherein the carbon atom with the free valence is amember of an aromatic ring. Aromatic groups include arylene groups andaromatic groups with more than two atoms removed. For the purposes ofthis application aromatic includes heterocycle. “Heterocycle” or“heteroaryl” means an aromatic group wherein 1 to 5 of the indicatedcarbon atoms are replaced by a heteroatom chosen from nitrogen, oxygen,sulfur, phosphorus, boron and silicon wherein the atom with the freevalence is a member of an aromatic ring, and any heterocyclic ketone andthioketone derivative thereof. Thus, heterocycle includes thienyl,furyl, pyrrolyl, pyrimidinyl, oxalyl, indolyl, purinyl, quinolyl,isoquinolyl, thiazolyl, imidozyl, etc.

Importantly, the Y aromatic groups of the conductive oligomer may bedifferent, i.e. the conductive oligomer may be a heterooligomer. Thatis, a conductive oligomer may comprise a oligomer of a single type of Ygroups, or of multiple types of Y groups.

The aromatic group may be substituted with a substitution group,generally depicted herein as R. R groups may be added as necessary toaffect the packing of the conductive oligomers, i.e. R groups may beused to alter the association of the oligomers in the monolayer. Rgroups may also be added to 1) alter the solubility of the oligomer orof compositions containing the oligomers; 2) alter the conjugation orelectrochemical potential of the system; and 3) alter the charge orcharacteristics at the surface of the monolayer.

In a preferred embodiment, when the conductive oligomer is greater thanthree subunits, R groups are preferred to increase solubility whensolution synthesis is done. However, the R groups, and their positions,are chosen to minimally effect the packing of the conductive oligomerson a surface, particularly within a monolayer, as described below. Ingeneral, only small R groups are used within the monolayer, with largerR groups generally above the surface of the monolayer. Thus for examplethe attachment of methyl groups to the portion of the conductiveoligomer within the monolayer to increase solubility is preferred, withattachment of longer alkoxy groups, for example, C3 to 010, ispreferably done above the monolayer surface. In general, for the systemsdescribed herein, this generally means that attachment of stericallysignificant R groups is not done on any of the first two or threeoligomer subunits, depending on the average length of the moleculesmaking up the monolayer.

Suitable R groups include, but are not limited to, hydrogen, alkyl,alcohol, aromatic, amino, amido, nitro, ethers, esters, aldehydes,sulfonyl, silicon moieties, halogens, sulfur containing moieties,phosphorus containing moieties, and ethylene glycols. In the structuresdepicted herein, R is hydrogen when the position is unsubstituted. Itshould be noted that some positions may allow two substitution groups, Rand R′, in which case the R and R′ groups may be either the same ordifferent.

By “alkyl group” or grammatical equivalents herein is meant a straightor branched chain alkyl group, with straight chain alkyl groups beingpreferred. If branched, it may be branched at one or more positions, andunless specified, at any position. The alkyl group may range from about1 to about 30 carbon atoms (C1-C30), with a preferred embodimentutilizing from about 1 to about 20 carbon atoms (C1-C20), with about C1through about C12 to about C15 being preferred, and C1 to C5 beingparticularly preferred, although in some embodiments the alkyl group maybe much larger. Also included within the definition of an alkyl groupare cycloalkyl groups such as C5 and C6 rings, and heterocyclic ringswith nitrogen, oxygen, sulfur or phosphorus. Alkyl also includesheteroalkyl, with heteroatoms of sulfur, oxygen, nitrogen, and siliconebeing preferred. Alkyl includes substituted alkyl groups. By“substituted alkyl group” herein is meant an alkyl group furthercomprising one or more substitution moieties “R”, as defined above.

By “amino groups” or grammatical equivalents herein is meant —NH₂, —NHRand —NR₂ groups, with R being as defined herein.

By “nitro group” herein is meant an —NO₂ group.

By “sulfur containing moieties” herein is meant compounds containingsulfur atoms, including but not limited to, thia-, thio- andsulfo-compounds, thiols (—SH and —SR), and sulfides (—RSR—). By“phosphorus containing moieties” herein is meant compounds containing \phosphorus, including, but not limited to, phosphines and phosphates. By“silicon containing moieties” herein is meant compounds containingsilicon.

By “ether” herein is meant an —O—R group. Preferred ethers includealkoxy groups, with —O—(CH₂)₂CH₃ and —O—(CH₂)₄CH₃ being preferred.

By “ester” herein is meant a —COOR group.

By “halogen” herein is meant bromine, iodine, chlorine, or fluorine.Preferred substituted alkyls are partially or fully halogenated alkylssuch as CF₃, etc.

By “aldehyde” herein is meant —RCHO groups.

By “alcohol” herein is meant —OH groups, and alkyl alcohols —ROH. By“amido” herein is meant —RCONH— or RCONR— groups.

By “ethylene glycol” or “(poly)ethylene glycol” herein is meant a—(O—CH₂—CH₂)_(n) group, although each carbon atom of the ethylene groupmay also be singly or doubly substituted, i.e. —(O—CR₂—CR₂)_(n)—, with Ras described above. Ethylene glycol derivatives with other heteroatomsin place of oxygen (i.e. —(N—CH₂—CH₂)_(n)— or —(S—CH₂—CH₂)_(n)—, or withsubstitution groups) are also preferred.

Preferred substitution groups include, but are not limited to, methyl,ethyl, propyl, alkoxy groups such as —O—(CH₂)₂CH₃ and —O—(CH₂)₄CH₃ andethylene glycol and derivatives thereof.

Preferred aromatic groups include, but are not limited to, phenyl,naphthyl, naphthalene, anthracene, phenanthroline, pyrole, pyridine,thiophene, porphyrins, and substituted derivatives of each of these,included fused ring derivatives.

In the conductive oligomers depicted herein, when g is 1, B-D is a bondlinking two atoms or chemical moieties. In a preferred embodiment, B-Dis a conjugated bond, containing overlapping or conjugated n-orbitals.

Preferred B-D bonds are selected from acetylene (—C≡C—, also calledalkyne or ethyne), alkene (—CH≡CH—, also called ethylene), substitutedalkene (—CR═CR—, —CH═CR— and —CR═CH—), amide (—NH—CO— and —NR—CO— or—CO—NH— and —CO—NR—), azo (—N═N—), esters and thioesters (—CO—O—,—O—CO—, —CS—O— and —O—CS—) and other conjugated bonds such as (—CH═N—,—CR═N—, —N═CH— and —N═CR—), (—SiH═SiH—, —SiR═SiH—, and —SiR═SiR—),(—SiH═CH—, —SiR═CH—, —SiH═CR—, —SIR═CR—, —CH═SiH—, —CH═SiR—, and—CR═SiR—). Particularly preferred B-D bonds are acetylene, alkene,amide, and substituted derivatives of these three, and azo. Especiallypreferred B-D bonds are acetylene, alkene and amide. The oligomercomponents attached to double bonds may be in the trans or cisconformation, or mixtures. Thus, either B or D may include carbon,nitrogen or silicon. The substitution groups are as defined as above forR.

When g=0 in the Structure 1 conductive oligomer, e is preferably 1 andthe D moiety may be carbonyl or a heteroatom moiety as defined above.

As above for the Y rings, within any single conductive oligomer, the B-Dbonds (or moieties, when g=0) may be all the same, or at least one maybe different. For example, when m is zero, the terminal B-D bond may bean amide bond, and the rest of the B-D bonds may be acetylene bonds.Generally, when amide bonds are present, as few amide bonds as possibleare preferable, but in some embodiments all the B-D bonds are amidebonds. Thus, as outlined above for the Y rings, one type of B-D bond maybe present in the conductive oligomer within a monolayer as describedbelow, and another type above the monolayer level, for example to givegreater flexibility for nucleic acid hybridization when the nucleic acidis attached via a conductive oligomer.

In the structures depicted herein, n is an integer from 1 to 50,although longer oligomers may also be used (see for example Schumm etal., Angew. Chem. Int. Ed. Engl. 1994 33(13):1360). Without being boundby theory, it appears that for efficient hybridization of nucleic acidson a surface, the hybridization should occur at a distance from thesurface, i.e. the kinetics of hybridization increase as a function ofthe distance from the surface, particularly for long oligonucleotides of200 to 300 basepairs. Accordingly, when a nucleic acid is attached via aconductive oligomer, as is more fully described below, the length of theconductive oligomer is such that the closest nucleotide of the nucleicacid is positioned from about 6 Å to about 100 Å (although distances ofup to 500 Å may be used) from the electrode surface, with from about 15Å to about 60 Å being preferred and from about 25 Å to about 60 Å alsobeing preferred. Accordingly, n will depend on the size of the aromaticgroup, but generally will be from about 1 to about 20, with from about 2to about 15 being preferred and from about 3 to about 10 beingespecially preferred.

In the structures depicted herein, m is either 0 or 1. That is, when mis 0, the conductive oligomer may terminate in the B-D bond or D moiety,i.e. the D atom is attached to the nucleic acid either directly or via alinker. In some embodiments, for example when the conductive oligomer isattached to a phosphate of the ribose-phosphate backbone of a nucleicacid, there may be additional atoms, such as a linker, attached betweenthe conductive oligomer and the nucleic acid. Additionally, as outlinedbelow, the D atom may be the nitrogen atom of the amino-modified ribose.Alternatively, when m is 1, the conductive oligomer may terminate in Y,an aromatic group, i.e. the aromatic group is attached to the nucleicacid or linker.

As will be appreciated by those in the art, a large number of possibleconductive oligomers may be utilized. These include conductive oligomersfalling within the Structure 1 and Structure 8 formulas, as well asother conductive oligomers, as are generally known in the art, includingfor example, compounds comprising fused aromatic rings or Teflon®-likeoligomers, such as —(CF₂)_(n), (CHF)_(n)— and —(CFR)_(n)—. See forexample, Schumm et al., Angew. Chem. Intl. Ed. Engl. 33:1361 (1994);Grosshenny et al., Platinum Metals Rev. 40(1):26-35 (1996); Tour, Chem.Rev. 96:537-553 (1996); Hsung et al., Organometallics 14:4808-4815(1995; and references cited therein, all of which are expresslyincorporated by reference.

Particularly preferred conductive oligomers of this embodiment aredepicted below:

Structure 2 is Structure 1 when g is 1. Preferred embodiments ofStructure 2 include: e is zero, Y is pyrole or substituted pyrole; e iszero, Y is thiophene or substituted thiophene; e is zero, Y is furan orsubstituted furan; e is zero, Y is phenyl or substituted phenyl; e iszero, Y is pyridine or substituted pyridine; e is 1, B-D is acetyleneand Y is phenyl or substituted phenyl (see Structure 4 below). Apreferred embodiment of Structure 2 is also when e is one, depicted asStructure 3 below:

Preferred embodiments of Structure 3 are: Y is phenyl or substitutedphenyl and B-D is azo; Y is phenyl or substituted phenyl and B-D isacetylene; Y is phenyl or substituted phenyl and B-D is alkene; Y ispyridine or substituted pyridine and B-D is acetylene; Y is thiophene orsubstituted thiophene and B-D is acetylene; Y Is furan or substitutedfuran and B-D is acetylene; Y is thiophene or furan (or substitutedthiophene or furan) and B-D are alternating alkene and acetylene bonds.

Most of the structures depicted herein utilize a Structure 3 conductiveoligomer. However, any. Structure 3 oligomers may be substituted withany of the other structures depicted herein, i.e. Structure 1 or 8oligomer, or other conducting oligomer, and the use of such Structure 3depiction is not meant to limit the scope of the invention.

Particularly preferred embodiments of Structure 3 include Structures 4,5, 6 and 7, depicted below:

Particularly preferred embodiments of Structure 4 include: n is two, mis one, and R is hydrogen; n is three, m is zero, and R is hydrogen; andthe use of R groups to increase solubility.

When the B-D bond is an amide bond, as in Structure 5, the conductiveoligomers are pseudopeptide oligomers. Although the amide bond inStructure 5 is depicted with the carbonyl to the left, i.e. —CONH—, thereverse may also be used, i.e. —NHCO—. Particularly preferredembodiments of Structure 5 include: n is two, m is one, and R ishydrogen; n is three, m is zero, and R is hydrogen (in this embodiment,the terminal nitrogen (the D atom) may be the nitrogen of theamino-modified ribose); and the use of R groups to increase solubility.

Preferred embodiments of Structure 6 include the first n is two, secondn is one, m is zero, and all R groups are hydrogen, or the use of Rgroups to increase solubility.

Preferred embodiments of Structure 7 include: the first n is three, thesecond n is from 1-3, with m being either 0 or 1, and the use of Rgroups to increase solubility.

In a preferred embodiment, the conductive oligomer has the structuredepicted in Structure 8:

In this embodiment, C are carbon atoms, n is an integer from 1 to 50, mis 0 or 1, J is a heteroatom selected from the group consisting ofoxygen, nitrogen, silicon, phosphorus, sulfur, carbonyl or sulfoxide,and G is a bond selected from alkane, alkene or acetylene, such thattogether with the two carbon atoms the C-G-C group is an alkene(—CH═CH—), substituted alkene (—CR═CR—) or mixtures thereof (—CH═CR— or—CR═CH—), acetylene (—C≡C—), or alkane (—CR₂—CR₂—, with R being eitherhydrogen or a substitution group as described herein). The G bond ofeach subunit may be the same or different than the G bonds of othersubunits; that is, alternating oligomers of alkene and acetylene bondscould be used, etc. However, when G is an alkane bond, the number ofalkane bonds in the oligomer should be kept to a minimum, with about sixor less sigma bonds per conductive oligomer being preferred. Alkenebonds are preferred, and are generally depicted herein, although alkaneand acetylene bonds may be substituted in any structure or embodimentdescribed herein as will be appreciated by those in the art.

In some embodiments, for example when ETMs are not present, if m=0 thenat least one of the G bonds is not an alkane bond.

In a preferred embodiment, the m of Structure 8 is zero. In aparticularly preferred embodiment, m is zero and G is an alkene bond, asis depicted in Structure 9:

The alkene oligomer of structure 9, and others depicted herein, aregenerally depicted in the preferred trans configuration, althougholigomers of cis or mixtures of trans and cis may also be used. Asabove, R groups may be added to alter the packing of the compositions onan electrode, the hydrophilicity or hydrophobicity of the oligomer, andthe flexibility, i.e. the rotational, torsional or longitudinalflexibility of the oligomer n is as defined above.

In a preferred embodiment, R is hydrogen, although R may be also alkylgroups and polyethylene glycols or derivatives.

In an alternative embodiment, the conductive oligomer may be a mixtureof different types of oligomers, for example of structures 1 and 8.

In addition, particularly for use with mechanism-2 systems, themonolayer comprises conductive oligomers, and the terminus of at leastsome of the conductive oligomers in the monolayer are electronicallyexposed. By “electronically exposed” herein is meant that upon theplacement of an ETM in close proximity to the terminus, and afterinitiation with the appropriate signal, a signal dependent on thepresence of the ETM may be detected. The conductive oligomers may or maynot have terminal groups. Thus, in a preferred embodiment, there is noadditional terminal group, and the conductive oligomer terminates withone of the groups depicted in Structures 1 to 9; for example, a BD bondsuch as an acetylene bond. Alternatively, in a preferred embodiment, aterminal group is added, sometimes depicted herein as “Q”. A terminalgroup may be used for several reasons; for example, to contribute to theelectronic availability of the conductive oligomer for detection ofETMs, or to alter the surface of the SAM for other reasons, for exampleto prevent non-specific binding. For example, when the target analyte isa nucleic acid, there may be negatively charged groups on the terminusto form a negatively charged surface such that when the nucleic acid isDNA or RNA the nucleic acid is repelled or prevented from lying down onthe surface, to facilitate hybridization. Preferred terminal groupsinclude —NH₂, —OH, —COOH, and alkyl groups such as —CH₃, and(poly)alkyloxides such as (poly)ethylene glycol, with —OCH₂CH₂OH,—(OCH₂CH₂O)₂H, —(OCH₂CH₂O)₃H, and —(OCH₂CH₂O)₄H being preferred.

In one embodiment, it is possible to use mixtures of conductiveoligomers with different types of terminal groups. Thus, for example,some of the terminal groups may facilitate detection, and some mayprevent non-specific binding.

It will be appreciated that the monolayer may comprise differentconductive oligomer species, although preferably the different speciesare chosen such that a reasonably uniform SAM can be formed. Thus, forexample, when capture binding ligands such as nucleic acids arecovalently attached to the electrode using conductive oligomers, it ispossible to have one type of conductive oligomer used to attach thenucleic acid, and another type functioning to detect the ETM. Similarly,it may be desirable to have mixtures of different lengths of conductiveoligomers in the monolayer, to help reduce nonspecific signals. Thus,for example, preferred embodiments utilize conductive oligomers thatterminate below the surface of the rest of the monolayer, i.e. below theinsulator layer, if used, or below some fraction of the other conductiveoligomers. Similarly, the use of different conductive oligomers may bedone to facilitate monolayer formation, or to make monolayers withaltered properties.

In a preferred embodiment, the monolayer may further comprise insulatormoieties. By “insulator” herein is meant a substantially nonconductingoligomer, preferably linear. By “substantially nonconducting” herein ismeant that the insulator will not transfer electrons at 100 Hz. The rateof electron transfer through the insulator is preferably slower than therate through the conductive oligomers described herein.

In a preferred embodiment, the insulators have a conductivity, S, ofabout 10⁻⁷Ω⁻¹ cm⁻¹ or lower, with less than about 10⁻⁸Ω⁻¹ cm⁻¹ beingpreferred. See generally Gardner et al., supra.

Generally, insulators are alkyl or heteroalkyl oligomers or moietieswith sigma bonds, although any particular insulator molecule may containaromatic groups or one or more conjugated bonds. By “heteroalkyl” hereinis meant an alkyl group that has at least one heteroatom, i.e. nitrogen,oxygen, sulfur, phosphorus, silicon or boron included in the chain.Alternatively, the insulator may be quite similar to a conductiveoligomer with the addition of one or more heteroatoms or bonds thatserve to inhibit or slow, preferably substantially, electron transfer.

Suitable insulators are known in the art, and include, but are notlimited to, —(CH₂)_(n)—, —(CRH)_(n)—, and —(CR₂)_(n)—, ethylene glycolor derivatives using other heteroatoms in place of oxygen, i.e. nitrogenor sulfur (sulfur derivatives are not preferred when the electrode isgold).

As for the conductive oligomers, the insulators may be substituted withR groups as defined herein to alter the packing of the moieties orconductive oligomers on an electrode, the hydrophilicity orhydrophobicity of the insulator, and the flexibility, i.e. therotational, torsional or longitudinal flexibility of the insulator. Forexample, branched alkyl groups may be used. Similarly, the insulatorsmay contain terminal groups, as outlined above, particularly toinfluence the surface of the monolayer.

The length of the species making up the monolayer will vary as needed.As outlined above, it appears that binding of target analytes (forexample, hybridization of nucleic acids) is more efficient at a distancefrom the surface. The species to which capture binding ligands areattached (as outlined below, these can be either insulators orconductive oligomers) may be basically the same length as the monolayerforming species or longer than them, resulting in the capture bindingligands being more accessible to the solvent for hybridization. In someembodiments, the conductive oligomers to which the capture bindingligands are attached may be shorter than the monolayer.

As will be appreciated by those in the art, the actual combinations andratios of the different species making up the monolayer can vary widely,and will depend on whether mechanism-1 or -2 is used, and whether a oneelectrode system or two electrode system is used, as is more fullyoutlined below. Generally, three component systems are preferred formechanism-2 systems, with the first species comprising a capture bindingligand containing species (termed a capture probe when the targetanalyte is a nucleic acid), attached to the electrode via either aninsulator or a conductive oligomer. The second species are theconductive oligomers, and the third species are insulators. In thisembodiment, the first species can comprise from about 90% to about 1%,with from about 20% to about 40% being preferred. When the targetanalytes are nucleic acids, from about 30% to about 40% is especiallypreferred for short oligonucleotide targets and from about 10% to about20% is preferred for longer targets. The second species can comprisefrom about 1% to about 90%, with from about 20% to about 90% beingpreferred, and from about 40% to about 60% being especially preferred.The third species can comprise from about 1% to about 90%, with fromabout 20% to about 40% being preferred, and from about 15% to about 30%being especially preferred. Preferred ratios of first:second:thirdspecies are 2:2:1 for short targets, 1:3:1 for longer targets, withtotal thiol concentration (when used to attach these species, as is morefully outlined below) in the 500 μM to 1 mM range, and 833 μM beingpreferred.

Alternatively, two component systems can be used. In one embodiment, foruse in either mechanism1 or mechanism-2 systems, the two components arethe first and second species. In this embodiment, the first species cancomprise from about 1% to about 90%, with from about 1% to about 40%being preferred, and from about 10% to about 40% being especiallypreferred. The second species can comprise from about 1% to about 90%,with from about 10% to about 60% being preferred, and from about 20% toabout 40% being especially preferred. Alternatively, for mechanism-1systems, the two components are the first and the third species. In thisembodiment, the first species can comprise from about 1% to about 90%,with from about 1% to about 40% being preferred, and from about 10% toabout 40% being especially preferred. The second species can comprisefrom about 1% to about 90%, with from about 10% to about 60% beingpreferred, and from about 20% to about 40% being especially preferred.

The covalent attachment of the conductive oligomers and insulators tothe electrode may be accomplished in a variety of ways, depending on theelectrode and the composition of the insulators and conductive oligomersused. In a preferred embodiment, the attachment linkers with covalentlyattached nucleosides or nucleic acids as depicted herein are covalentlyattached to an electrode. Thus, one end or terminus of the attachmentlinker is attached to the nucleoside or nucleic acid, and the other isattached to an electrode. In some embodiments it may be desirable tohave the attachment linker attached at a position other than a terminus,or even to have a branched attachment linker that is attached to anelectrode at one terminus and to two or more nucleosides at othertermini, although this is not preferred. Similarly, the attachmentlinker may be attached at two sites to the electrode, as is generallydepicted in Structures 11-13. Generally, some type of linker is used, asdepicted below as “A” in Structure 10, where “X” is the conductiveoligomer, “I” is an insulator and the hatched surface is the electrode:

In this embodiment, A is a linker or atom. The choice of “A” will dependin part on the characteristics of the electrode. Thus, for example, Amay be a sulfur moiety when a gold electrode is used. Alternatively,when metal oxide electrodes are used, A may be a silicon (silane) moietyattached to the oxygen of the oxide (see for example Chen et al.,Langmuir 10:3332-3337 (1994); Lenhard et al., J. Electroanal. Chem.78:195-201 (1977), both of which are expressly incorporated byreference). When carbon based electrodes are used, A may be an aminomoiety (preferably a primary amine; see for example Deinhammer et al.,Langmuir 10:1306-1313 (1994)). Thus, preferred A moieties include, butare not limited to, silane moieties, sulfur moieties (including alkylsulfur moieties), and amino moieties. In a preferred embodiment, epoxidetype linkages with redox polymers such as are known in the art are notused.

Although depicted herein as a single moiety, the insulators andconductive oligomers may be attached to the electrode with more than one“A” moiety; the “A” moieties may be the same or different. Thus, forexample, when the electrode is a gold electrode, and “A” is a sulfuratom or moiety, multiple sulfur atoms may be used to attach theconductive oligomer to the electrode, such as is generally depictedbelow in Structures 11, 12 and 13. As will be appreciated by those inthe art, other such structures can be made. In Structures 11, 12 and 13,the A moiety is just a sulfur atom, but substituted sulfur moieties mayalso be used.

It should also be noted that similar to Structure 13, it may be possibleto have a conductive oligomer terminating in a single carbon atom withthree sulfur moities attached to the electrode. Additionally, althoughnot always depicted herein, the conductive oligomers and insulators mayalso comprise a “Q” terminal group.

In a preferred embodiment, the electrode is a gold electrode, andattachment is via a sulfur linkage as is well known in the art, i.e. theA moiety is a sulfur atom or moiety. Although the exact characteristicsof the gold-sulfur attachment are not known, this linkage is consideredcovalent for the purposes of this invention. A representative structureis depicted in Structure 14, using the Structure 3 conductive oligomer,although as for all the structures depicted herein, any of theconductive oligomers, or combinations of conductive oligomers, may beused. Similarly, any of the conductive oligomers or insulators may alsocomprise terminal groups as described herein. Structure 14 depicts the“A” linker as comprising just a sulfur atom, although additional atomsmay be present (i.e. linkers from the sulfur to the conductive oligomeror substitution groups). In addition, Structure 14 shows the sulfur atomattached to the Y aromatic group, but as will be appreciated by those inthe art, it may be attached to the B-D group (i.e. an acetylene) aswell.

In general, thiol linkages are preferred when either two sets ofelectrodes are used (i.e. the detection electrodes comprising the SAMsare not used at high electrophoretic voltages (i.e. greater than 800 or900 mV), that can cause oxidation of the thiol linkage and thus loss ofthe SAM), or, if one set of electrodes is used, lower electrophoreticvoltages are used as is generally described below.

In a preferred embodiment, the electrode is a carbon electrode, i.e. aglassy carbon electrode, and attachment is via a nitrogen of an aminegroup. A representative structure is depicted in Structure 15. Again,additional atoms may be present, i.e. Z type linkers and/or terminalgroups.

In Structure 16, the oxygen atom is from the oxide of the metal oxideelectrode. The Si atom may also contain other atoms, i.e. be a siliconmoiety containing substitution groups. Other attachments for SAMs toother electrodes are known in the art; see for example Napier et al.,Langmuir, 1997, for attachment to indium tin oxide electrodes, and alsothe chemisorption of phosphates to an indium tin oxide electrode (talkby H. Holden Thorpe, CHI conference, May 4-5, 1998).

In a preferred embodiment, the detection electrode further comprises acapture binding ligand, preferably covalently attached. In general, formost of the “mechanism-2” embodiments described herein, there are atleast two binding ligands used per target analyte molecule; a “capture”or “anchor” binding ligand (also referred to herein as a “captureprobe”, particularly in reference to a nucleic acid binding ligand) thatis attached to the detection electrode as described herein, and asoluble binding ligand, that binds independently to the target analyte,and either directly or indirectly comprises at least one ETM.

Thus, in preferred embodiments, although it is not required, the targetsequences are immobilized on the electrode surface. This is preferablydone using capture probes and optionally one or more capture extenderprobes. When only capture probes are utilized, it is necessary to haveunique capture probes for each target sequence; that is, the surfacemust be customized to contain unique capture probes. Alternatively,capture extender probes may be used, that allow a “universal” surface,i.e. a surface containing a single type of capture probe that can beused to detect any target sequence. “Capture extender” probes have afirst portion that will hybridize to all or part of the capture probe,and a second portion that will hybridize to a first portion of thetarget sequence. This then allows the generation of customized solubleprobes, which as will be appreciated by those in the art is generallysimpler and less costly. As shown herein, two capture extender probesmay be used. This has generally been done to stabilize assay complexes(for example when the target sequence is large, or when large amplifierprobes (particularly branched or dendrimer amplifier probes) are used.

In a preferred embodiment, the nucleic acids are added after theformation of the SAM, discussed herein. This may be done in a variety ofways, as will be appreciated by those in the art. In one embodiment,conductive oligomers with terminal functional groups are made, withpreferred embodiments utilizing activated carboxylates andisothiocyanates, that will react with primary amines that are put ontothe nucleic acid using an activated carboxylate. These two reagents havethe advantage of being stable in aqueous solution, yet react withprimary alkylamines. However, the primary aromatic amines and secondaryand tertiary amines of the bases should not react, thus allowing sitespecific addition of nucleic acids to the surface. This allows thespotting of probes (either capture or detection probes, or both) usingknown methods (ink jet, spotting, etc.) onto the surface.

In addition, there are a number of non-nucleic acid methods that can beused to immobilize a nucleic acid on a surface. For example, bindingpartner pairs can be utilized; i.e. one binding partner is attached tothe terminus of an attachment linker, described below, and the other tothe end of the nucleic acid. This may also be done without using anucleic acid capture probe; that is, one binding partner serves as thecapture probe and the other is attached to either the target sequence ora capture extender probe. That is, either the target sequence comprisesthe binding partner, or a capture extender probe that will hybridize tothe target sequence comprises the binding partner. Suitable bindingpartner pairs include, but are not limited to, hapten pairs such asbiotin/streptavidin; antigens/antibodies; NTA/histidine tags; etc. Ingeneral, smaller binding partners are preferred, such that the electronscan pass from the nucleic acid into the conductive oligomer to allowdetection.

In a preferred embodiment, when the target sequence itself is modifiedto contain a binding partner, the binding partner is attached via amodified nucleotide that can be enzymatically attached to the targetsequence, for example during a PCR target amplification step.Alternatively, the binding partner should be easily attached to thetarget sequence.

Alternatively, a capture extender probe may be utilized that has anucleic acid portion for hybridization to the target as well as abinding partner (for example, the capture extender probe may comprise anon-nucleic acid portion such as an alkyl linker that is used to attacha binding partner). In this embodiment, it may be desirable tocross-link the double-stranded nucleic acid of the target and captureextender probe for stability, for example using psoralen as is known inthe art.

In one embodiment, the target is not bound to the electrode surfaceusing capture probes. In this embodiment, what is important, as for allthe assays herein, is that excess label probes be removed prior todetection and that the assay complex be in proximity to the surface. Aswill be appreciated by those in the art, this may be accomplished inother ways. For example, the assay complex comprising the ETMs may bepresent on beads that are added to the electrode comprising themonolayer, and then the beads brought into proximity of the electrodesurface using techniques well known in the art, including gravitysettling of the beads on the surface, electrostatic or magneticinteractions between bead components and the surface, using bindingpartner attachment as outlined above. Alternatively, after the removalof excess reagents such as excess label probes, the assay complex may bedriven down to the surface, for example by pulsing the system with avoltage sufficient to drive the assay complex to the surface.

However, preferred embodiments utilize assay complexes attached vianucleic acid capture probes.

Generally, the capture binding ligand allows the attachment of a targetanalyte to the detection electrode, for the purposes of detection. As ismore fully outlined below, attachment of the target analyte to thecapture binding ligand may be direct (i.e. the target analyte binds tothe capture binding ligand) or indirect (one or more capture extenderligands may be used).

The method of attachment of the capture binding ligands to theattachment linker (either an insulator or conductive oligomer) willgenerally be done as is known in the art, and will depend on both thecomposition of the attachment linker and the capture binding ligand. Ingeneral, the capture binding ligands are attached to the attachmentlinker through the use of functional groups on each that can then beused for attachment. Preferred functional groups for attachment areamino groups, carboxy groups, oxo groups and thiol groups. Thesefunctional groups can then be attached, either directly or indirectlythrough the use of a linker, sometimes depicted herein as “Z”. Linkersare well known in the art; for example, homo- or hetero-bifunctionallinkers as are well known (see 1994 Pierce Chemical Company catalog,technical section on cross-linkers, pages 155-200, incorporated hereinby reference). Preferred Z linkers include, but are not limited to,alkyl groups (including substituted alkyl groups and alkyl groupscontaining heteroatom moieties), with short alkyl groups, esters, amide,amine, epoxy groups and ethylene glycol and derivatives being preferred,with propyl, acetylene, and C2 alkene being especially preferred. Z mayalso be a sulfone group, forming sulfonamide linkages.

In this way, capture binding ligands comprising proteins, lectins,nucleic acids, small organic molecules, carbohydrates, etc. can beadded.

A preferred embodiment utilizes proteinaceous capture binding ligands.As is known in the art, any number of techniques may be used to attach aproteinaceous capture binding ligand to an attachment linker. “Protein”as used herein includes proteins, polypeptides, and peptides. Theprotein may be made up of naturally occurring amino acids and peptidebonds, or synthetic peptidomimetic structures. The side chains may be ineither the (R) or the (S) configuration. In the preferred embodiment,the amino acids are in the (S) or L-configuration. If non-naturallyoccurring side chains are used, non-amino acid substituents may be used,for example to prevent or retard in vivo degradations. A wide variety oftechniques are known to add moieties to proteins.

A preferred embodiment utilizes nucleic acids as the capture bindingligand. As will be appreciated by those in the art, many of thetechniques outlined below apply in a similar manner to non-nucleic acidsystems.

The capture probe nucleic acid is covalently attached to the electrode,via an “attachment linker”, that can be either a conductive oligomer(required for mechanism-1 systems) or an insulator. By “covalentlyattached” herein is meant that two moieties are attached by at least onebond, including sigma bonds, pi bonds and coordination bonds.

Thus, one end of the attachment linker is attached to a nucleic acid (orother binding ligand), and the other end (although as will beappreciated by those in the art, it need not be the exact terminus foreither) is attached to the electrode. Thus, any of structures depictedherein may further comprise a nucleic acid effectively as a terminalgroup. Thus, the present invention provides compositions comprisingnucleic acids covalently attached to electrodes as is generally depictedbelow in Structure 17:

In Structure 17, the hatched marks on the left represent an electrode. Xis a conductive oligomer and I is an insulator as defined herein. F, isa linkage that allows the covalent attachment of the electrode and theconductive oligomer or insulator, including bonds, atoms or linkers suchas is described herein, for example as “A”, defined below. F₂ is alinkage that allows the covalent attachment of the conductive oligomeror insulator to the nucleic acid, and may be a bond, an atom or alinkage as is herein described. F₂ may be part of the conductiveoligomer, part of the insulator, part of the nucleic acid, or exogeneousto both, for example, as defined herein for “Z”.

In a preferred embodiment, the capture probe nucleic acid is covalentlyattached to the electrode via a conductive oligomer. The covalentattachment of the nucleic acid and the conductive oligomer may bEaccomplished in several ways. In a preferred embodiment, the attachmentis via attachment to the base of the nucleoside, via attachment to thebackbone of the nucleic acid (either the ribose, the phosphate, or to ananalogous group of a nucleic acid analog backbone), or via a transitionmetal ligand, as described below. The techniques outlined below aregenerally described for naturally occurring nucleic acids, although aswill be appreciated by those in the art, similar techniques may be usedwith nucleic acid analogs, and in some cases with other binding ligands.

In a preferred embodiment, the conductive oligomer is attached to thebase of a nucleoside of the nucleic acid. This may be done in severalways, depending on the oligomer, as is described below. Ir oneembodiment, the oligomer is attached to a terminal nucleoside, i.e.either the 3′ or 5′ nucleoside of the nucleic acid. Alternatively, theconductive oligomer is attached to an internal nucleoside.

The point of attachment to the base will vary with the base. Generally,attachment at any position is possible. In some embodiments, for examplewhen the probe containing the ETMs may be used for hybridization (i.e.mechanism-1 systems), it is preferred to attach at positions notinvolved in hydroge bonding to the complementary base. Thus, forexample, generally attachment is to the 5 or 6 position of pyrimidinessuch as uridine, cytosine and thymine. For purines such as adenine andguanine, the linkage is preferably via the 8 position. Attachment tonon-standard bases is preferably done at the comparable positions.

In one embodiment, the attachment is direct; that is, there are nointervening atoms between the conductive oligomer and the base. In thisembodiment, for example, conductive oligomers with terminal acetylenebonds are attached directly to the base. Structure 18 is an example ofthis linkage, using a Structure 3 conductive oligomer and uridine as thebase, although other bases and conductive oligomers can be used as willbe appreciated by those in the art:

It should be noted that the pentose structures depicted herein may havehydrogen, hydroxy, phosphates or other groups such as amino groupsattached. In addition, the pentose and nucleoside structures depictedherein are depicted non-conventionally, as mirror images of the normalrendering. In addition, the pentose and nucleoside structures may alsocontain additional groups, such as protecting groups, at any position,for example as needed during synthesis.

In addition, the base may contain additional modifications as needed,i.e. the carbonyl or amine group may be altered or protected, forexample as is depicted in FIG. 3 or 10.

In an alternative embodiment, the attachment is any number of differentZ linkers, including amide anc amine linkages, as is generally depictedin Structure 19 using uridine as the base and a Structure 3 oligomer:

In this embodiment, Z is a linker. Preferably, Z is a short linker ofabout Ito about 10 atoms, with fro 1 to 5 atoms being preferred, thatmay or may not contain alkene, alkynyl, amine, amide, azo, imine, etc.,bonds. Linkers are known in the art; for example, homo- orhetero-bifunctional linkers as are well known (see 1994 Pierce ChemicalCompany catalog, technical section on cross-linkers, pages 155-200,incorporated herein by reference). Preferred Z linkers include, but arenot limited to, alkyl groups (including substituted alkyl groups andalkyl groups containing heteroatom moieties), with short alkyl groups,esters, amide, amine, epoxy groups and ethylene glycol and derivativesbeing preferred, with propyl, acetylene, and C₂ alkene being especiallypreferred. Z may also be a sulfone group, forming sulfonamide linkagesas discussed below.

In a preferred embodiment, the attachment of the nucleic acid and theconductive oligomer is done via attachment to the backbone of thenucleic acid. This may be done in a number of ways, including attachmentto a ribose of the ribose-phosphate backbone, or to the phosphate of thebackbone, or other groups of analogous backbones.

As a preliminary matter, it should be understood that the site ofattachment in this embodiment may be to a 3′ or 5′ terminal nucleotide,or to an internal nucleotide, as is more fully described below.

In a preferred embodiment, the conductive oligomer is attached to theribose of the ribose-phosphate backbone. This may be done in severalways. As is known in the art, nucleosides that are modified at eitherthe 2′ or 3′ position of the ribose with amino groups, sulfur groups,silicone groups, phosphorus groups, or oxo groups can be made (Imazawaet al., J. Org. Chem., 44:2039 (1979); Hobbs et al., J. Org. Chem.42(4):714 (1977); Verheyden et al., J. Orrg. Chem. 36(2):250 (1971);McGee et al., J. Org. Chem. 61; 781-785 (1996); Mikhailopulo et al.,Liebigs. Ann. Chem. 513-519 (1993); McGee et al., Nucleosides &Nucleotides 14(6):1329 (1995), all of which are incorporated byreference). These modified nucleosides are then used to add theconductive oligomers.

A preferred embodiment utilizes amino-modified nucleosides. Theseamino-modified riboses can then be used to form either amide or aminelinkages to the conductive oligomers. In a preferred embodiment, theamino group is attached directly to the ribose, although as will beappreciated by those in the art, short linkers such as those describedherein for “Z” may be present between the amino group and the ribose.

In a preferred embodiment, an amide linkage is used for attachment tothe ribose. Preferably, if the conductive oligomer of Structures 1-3 isused, m is zero and thus the conductive oligomer terminates in the amidebond. In this embodiment, the nitrogen of the amino group of theamino-modified ribose is the “D” atom of the conductive oligomer. Thus,a preferred attachment of this embodiment is depicted in Structure 20(using the Structure 3 conductive oligomer):

As will be appreciated by those in the art, Structure 20 has theterminal bond fixed as an amide bond.

In a preferred embodiment, a heteroatom linkage is used, i.e. oxo,amine, sulfur, etc. A preferred embodiment utilizes an amine linkage.Again, as outlined above for the amide linkages, for amine linkages, thenitrogen of the amino-modified ribose may be the “D” atom of theconductive oligomer when the Structure 3 conductive oligomer is used.Thus, for example, Structures 21 and 22 depict nucleosides with theStructures 3 and 9 conductive oligomers, respectively, using thenitrogen as the heteroatom, athough other heteroatoms can be used:

In Structure 21, preferably both m and t are not zero. A preferred Zhere is a methylene group, or other aliphatic alkyl linkers. One, two orthree carbons in this position are particularly useful for syntheticreasons.

In Structure 22, Z is as defined above. Suitable linkers includemethylene and ethylene.

In an alternative embodiment, the conductive oligomer is covalentlyattached to the nucleic acid via the phosphate of the ribose-phosphatebackbone (or analog) of a nucleic acid. In this embodiment, theattachment is direct, utilizes a linker or via an amide bond. Structure23 depicts a direct linkage, and Structure 24 depicts linkage via anamide bond (both utilize the Structure 3 conductive oligomer, althoughStructure 8 conductive oligomers are also possible). Structures 23 and24 depict the conductive oligomer in the 3′ position, although the 5′position is also possible. Furthermore, both Structures 23 and 24 depictnaturally occurring phosphodiester bonds, although as those in the artwill appreciate, non-standard analogs of phosphodiester bonds may alsobe used.

In Structure 23, if the terminal Y is present (i.e. m=1), thenpreferably Z is not present (i.e. t=0). If the terminal Y is notpresent, then Z is preferably present.

Structure 24 depicts a preferred embodiment, wherein the terminal B-Dbond is an amide bond, the terminal Y is not present, and Z is a linker,as defined herein.

In a preferred embodiment, the conductive oligomer is covalentlyattached to the nucleic acid via a transition metal ligand. In thisembodiment, the conductive oligomer is covalently attached to a ligandwhich provides one or more of the coordination atoms for a transitionmetal. In one embodiment, the ligand to which the conductive oligomer isattached also has the nucleic acid attached, as is generally depictedbelow in Structure 25. Alternatively, the conductive oligomer isattached to one ligand, and the nucleic acid is attached to anotherligand, as is generally depicted below in Structure 26. Thus, in thepresence of the transition metal, the conductive oligomer is covalentlyattached to the nucleic acid. Both of these structures depict Structure3 conductive oligomers, although other oligomers may be utilized.Structures 25 and 26 depict two representative structures:

In the structures depicted herein, M is a metal atom, with transitionmetals being preferred. Suitable transition metals for use in theinvention include, but are not limited to, cadmium (Cd), copper (Cu),cobalt (Co), palladium (Pd), zinc (Zn), iron (Fe), ruthenium (Ru),rhodium (Rh), osmium (Os), rhenium (Re), platinium (Pt), scandium (Sc),titanium (Ti), Vanadium (V), chromium (Cr), manganese (Mn), nickel (Ni),Molybdenum (Mo), technetium (Tc), tungsten (W), and iridium (Ir). Thatis, the first series of transition metals, the platinum metals (Ru, Rh,Pd, Os, Ir and Pt), along with Fe, Re, W, Mo and Tc, are preferred.Particularly preferred are ruthenium, rhenium, osmium, platinium, cobaltand iron.

L are the co-ligands, that provide the coordination atoms for thebinding of the metal ion. As will be appreciated by those in the art,the number and nature of the co-ligands will depend on the coordinationnumber of the metal ion. Mono-, di- or polydentate co-ligands may beused at any position. Thus, for example, when the metal has acoordination number of six, the L from the terminus of the conductiveoligomer, the L contributed from the nucleic acid, and r, add up to six.Thus, when the metal has a coordination number of six, r may range fromzero (when all coordination atoms are provided by the other two ligands)to four, when all the co-ligands are monodentate. Thus generally, r willbe from 0 to 8, depending on the coordination number of the metal ionand the choice of the other ligands.

In one embodiment, the metal ion has a coordination number of six andboth the ligand attached to the conductive oligomer and the ligandattached to the nucleic acid are at least bidentate; that is, r ispreferably zero, one (i.e. the remaining co-ligand is bidentate) or two(two monodentate co-ligands are used).

As will be appreciated in the art, the co-ligands can be the same ordifferent. Suitable ligands fall into two categories: ligands which usenitrogen, oxygen, sulfur, carbon or phosphorus atoms (depending on themetal ion) as the coordination atoms (generally referred to in theliterature as sigma (a) donors) and organometallic ligands such asmetallocene ligands (generally referred to in the literature as pi (n)donors, and depicted herein as L_(m)). Suitable nitrogen donatingligands are well known in the art and include, but are not limited to,NH₂; NHR; NRR′; pyridine; pyrazine; isonicotinamide; imidazole;bipyridine and substituted derivatives of bipyridine; terpyridine andsubstituted derivatives; phenanthrolines, particularly1,10-phenanthroline (abbreviated phen) and substituted derivatives ofphenanthrolines such as 4,7-dimethylphenanthroline anddipyridol[3,2-a:2′,3′-c]phenazine (abbreviated dppz); dipyridophenazine;1,4,5,8,9,12-hexaazatriphenylene (abbreviated hat);9,10-phenanthrenequinone diimine (abbreviated phi);1,4,5,8-tetraazaphenanthrene (abbreviated tap);1,4,8,11-tetra-azacyclotetradecane (abbreviated cyclam), EDTA, EGTA andisocyanide. Substituted derivatives, including fused derivatives, mayalso be used. In some embodiments, porphyrins and substitutedderivatives of the porphyrin family may be used. See for example,Comprehensive Coordination Chemistry, Ed. Wilkinson et al., PergammonPress, 1987, Chapters 13.2 (pp 73-98), 21.1 (pp. 813-898) and 21.3 (pp915-957), alt of which are hereby expressly incorporated by reference.

Suitable sigma donating ligands using carbon, oxygen, sulfur andphosphorus are known in the art. For example, suitable sigma carbondonors are found in Cotton and Wilkenson, Advanced Organic Chemistry,5th Edition, John Wiley & Sons, 1988, hereby incorporated by reference;see page 38, for example. Similarly, suitable oxygen ligands includecrown ethers, water and others known in the art. Phosphines andsubstituted phosphines are also suitable; see page 38 of Cotton andWilkenson.

The oxygen, sulfur, phosphorus and nitrogen-donating ligands areattached in such a manner as to allow the heteroatoms to serve ascoordination atoms.

In a preferred embodiment, organometallic ligands are used. In additionto purely organic compounds for use as redox moieties, and varioustransition metal coordination complexes with δ-bonded organic ligandwith donor atoms as heterocyclic or exocyclic substituents, there isavailable a wide variety of transition metal organometallic compoundswith n-bonded organic ligands (see Advanced Inorganic Chemistry, 5thEd., Cotton & Wilkinson, John Wiley & Sons, 1988, chapter 26;Organometallics, A Concise Introduction, Elschenbroich et al., 2nd Ed.,1992, VCH; and Comprehensive Organometallic Chemistry II, A Review ofthe Literature 1982-1994, Abel et al. Ed., Vol. 7, chapters 7, 8, 10 &11, Pergamon Press, hereby expressly incorporated by reference). Suchorganometallic ligands include cyclic aromatic compounds such as thecyclopentadienide ion (C₅H₅(−1)] and various ring substituted and ringfused derivatives, such as the indenylide (−1) ion, that yield a classof bis(cyclopentadieyl) metal compounds, (i.e. the metallocenes); seefor example Robins et al., J. Am. Chem. Soc. 104:1882-1893 (1982); andGassman et al., J. Am. Chem. Soc. 108:4228-4229 (1986), incorporated byreference. Of these, ferrocene [(C₅H₅)₂Fe] and its derivatives areprototypical examples which have been used in a wide variety of chemical(Connelly at al., Chem. Rev. 96:877910 (1996), incorporated byreference) and electrochemical (Geiger at al., Advances inOrganometallic Chemistry 23:1-93; and Geiger at al., Advances inOrganometallic Chemistry 24:87, incorporated by reference) electrontransfer or “redox” reactions. Metallocene derivatives of a variety ofthe first, second and third row transition metals are potentialcandidates as redox moieties that are covalently attached to either theribose ring or the nucleoside base of nucleic acid. Other potentiallysuitable organometallic ligands include cyclic arenes such as benzene,to yield bis(arene)metal compounds and their ring substituted and ringfused derivatives, of which bis(benzene)chromium is a prototypicalexample, Other acyclic rr-bonded ligands such as the allyl(−1) ion, orbutadiene yield potentially suitable organometallic compounds, and allsuch ligands, in conjuction with other n-bonded and 5bonded ligandsconstitute the general class of organometallic compounds in which thereis a metal to carbon bond. Electrochemical studies of various dimers andoligomers of such compounds with bridging organic ligands, andadditional non-bridging ligands, as well as with and without metal-metalbonds are potential candidate redox moieties in nucleic acid analysis.

When one or more of the co-ligands is an organometallic ligand, theligand is generally attached via one of the carbon atoms of theorganometallic ligand, although attachment may be via other atoms forheterocyclic ligands. Preferred organometallic ligands includemetallocene ligands, including substituted derivatives and themetalloceneophanes (see page 1174 of Cotton and Wilkenson, supra). Forexample, derivatives of metallocene ligands such asmethylcyclopentadienyl, with multiple methyl groups being preferred,such as pentamethylcyclopentadienyl, can be used to increase thestability of the metallocene. In a preferred embodiment, only one of thetwo metallocene ligands of a metallocene are derivatized.

As described herein, any combination of ligands may be used. Preferredcombinations include: a) all ligands are nitrogen donating ligands; b)all ligands are organometallic ligands; and c) the ligand at theterminus of the conductive oligomer is a metallocene ligand and theligand provided by the nucleic acid is a nitrogen donating ligand, withthe other ligands, if needed, are either nitrogen donating ligands ormetallocene ligands, or a mixture. These combinations are depicted inrepresentative structures using the conductive oligomer of Structure 3are depicted in Structures 27 (using phenanthroline and amino asrepresentative ligands), 28 (using ferrocene as the metal-ligandcombination) and 29 (using cyclopentadienyl and amino as representativeligands).

In a preferred embodiment, the ligands used in the invention showaltered fluoroscent properties depending on the redox state of thechelated metal ion. As described below, this thus serves as anadditional mode of detection of electron transfer between the ETM andthe electrode.

In a preferred embodiment, as is described more fully below, the ligandattached to the nucleic acid is an amino group attached to the 2′ or 3′position of a ribose of the ribose-phosphate backbone. This ligand maycontain a multiplicity of amino groups so as to form a polydentateligand which binds the metal ion. Other preferred ligands includecyclopentadiene and phenanthroline.

The use of metal ions to connect the nucleic acids can serve as aninternal control or calibration of the system, to evaluate the number ofavailable nucleic acids on the surface. However, as will be appreciatedby those in the art, if metal ions are used to connect the nucleic acidsto the conductive oligomers, it is generally desirable to have thismetal ion complex have a different redox potential than that of the ETMsused in the rest of the system, as described below. This is generallytrue so as to be able to distinguish the presence of the capture probefrom the presence of the target sequence. This may be useful foridentification, calibration and/or quantification. Thus, the amount ofcapture probe on an electrode may be compared to the amount ofhybridized double stranded nucleic acid to quantify the amount of targetsequence in a sample. This is quite significant to serve as an internalcontrol of the sensor or system. This allows a measurement either priorto the addition of target or after, on the same molecules that will beused for detection, rather than rely on a similar but different controlsystem. Thus, the actual molecules that will be used for the detectioncan be quantified prior to any experiment. This is a significantadvantage over prior methods.

In a preferred embodiment, the capture probe nucleic acids (or otherbinding ligands) are covalently attached to the electrode via aninsulator. The attachment of nucleic acids (and other binding ligands)to insulators such as alkyl groups is well known, and can be done to thebase or the backbone, including the ribose or phosphate for backbonescontaining these moieties, or to alternate backbones for nucleic acidanalogs.

In a preferred embodiment, there may be one or more different captureprobe species on the surface. In some embodiments, there may be one typeof capture probe, or one type of capture probe extender, as is morefully described below. Alternatively, different capture probes, or onecapture probes with a multiplicity of different capture extender probescan be used. Similarly, it may be desirable (particular in the case ofnucleic acid analytes and binding ligands in mechanism-2 systems) to useauxiliary capture probes that comprise relatively short probe sequences,that can be used to “tack down” components of the system, for examplethe recruitment linkers, to increase the concentration of ETMs at thesurface.

Thus the present invention provides substrates comprising at least onedetection electrode comprising monolayers and capture binding ligands,useful in target analyte detection systems.

In a preferred embodiment, the compositions further comprise a solutionor soluble binding ligand, although as is more fully described below,for mechanism-1 systems, the ETMs may be added in the form ofnon-covalently attached hybridization indicators. Solution bindingligands are similar to capture binding ligands, in that they bind,preferably specifically, to target analytes. The solution binding ligandmay be the same or different from the capture binding ligand. Generally,the solution binding ligands are not directed attached to the surface,although they may be. The solution binding ligand either directlycomprises a recruitment linker that comprises at least one ETM or therecruitment linker binds, either directly or indirectly to the solutionbinding ligand.

Thus, “solution binding ligands” or “soluble binding ligands” or “signalcarriers” or “label probes” or “label binding ligands” with recruitmentlinkers comprising covalently attached ETMs are provided. That is, oneportion of the label probe or solution binding ligand directly orindirectly binds to the target analyte, and one portion comprises arecruitment linker comprising covalently attached ETMs. In some systems,for example in mechanism-1 nucleic acid systems, these may be the same.Similarly, for mechanism-1 systems, the recruitment linker comprisesnucleic acid that will hybridize to detection probes. The terms“electron donor moiety”, “electron acceptor moiety”, and “ETMs” (ETMs)or grammatical equivalents herein refers to molecules capable ofelectron transfer under certain conditions. It is to be understood thatelectron donor and acceptor capabilities are relative; that is, amolecule which can lose an electron under certain experimentalconditions will be able to accept an electron under differentexperimental conditions. It is to be understood that the number ofpossible electron donor moieties and electron acceptor moieties is verylarge, and that one skilled in the art of electron transfer compoundswill be able to utilize a number of compounds in the present invention.Preferred ETMs include, but are not limited to, transition metalcomplexes, organic ETMs, and electrodes.

In a preferred embodiment, the ETMs are transition metal complexes.Transition metals are those whose atoms have a partial or complete dshell of electrons. Suitable transition metals for use in the Inventionare listed above.

The transition metals are complexed with a variety of ligands, L,defined above, to form suitable transition metal complexes, as is wellknown in the art.

In addition to transition metal complexes, other organic electron donorsand acceptors may be covalently attached to the nucleic acid for use inthe invention. These organic molecules include, but are not limited to,riboflavin, xanthene dyes, azine dyes, acridine orange,N,N′-dimethyl-2,7diazapyrenium dichloride (DAP²⁺), methylviologen,ethidium bromide, quinones such as N,N′dimethylanthra(2,1,9-def:6,5,10-def:)diisoquinoline dichloride (ADIQ²⁺);porphyrins ([meso-tetrakis(N-methyl-x-pyridinium)porphyrintetrachloride], varlamine blue B hydrochloride, Bindschedler's green;2,6-dichloroindophenol, 2,6-dibromophenolindophenol; Brilliant crestblue (3-amino-9-dimethyl-amino-10-methylphenoxyazine chloride),methylene blue; Nile blue A (aminoaphthodiethylaminophenoxazinesulfate), indigo-5,5′,7,7′-tetrasulfonic acid, indigo-5,5′,7-trisulfonicacid; phenosafranine, indigo-5-monosulfonic acid; safranine T;bis(dimethylglyoximato)-iron(II) chloride; induline scarlet, neutralred, anthracene, coronene, pyrene, 9-phenylanthracene, rubrene,binaphthyl, DPA, phenothiazene, fluoranthene, phenanthrene, chrysene,1,8-diphenyl-1,3,5,7-octatetracene, naphthalene, acenaphthalene,perylene, TMPD and analogs and subsitituted derivatives of thesecompounds.

In one embodiment, the electron donors and acceptors are redox proteinsas are known in the art. However, redox proteins in many embodiments arenot preferred.

The choice of the specific ETMs will be influenced by the type ofelectron transfer detection used, as is generally outlined below.Preferred ETMs are metallocenes, with ferrocene being particularlypreferred.

In a preferred embodiment, a plurality of ETMs are used. As is shown inthe examples, the use of multiple ETMs provides signal amplification andthus allows more sensitive detection limits. As discussed below, whilethe use of multiple ETMs on nucleic acids that hybridize tocomplementary strands can cause decreases in T_(m)s of the hybridizationcomplexes depending on the number, site of attachment and spacingbetween the multiple ETMs, this is not a factor when the ETMs are on therecruitment linker (i.e. “mechanism-2” systems), since this does nothybridize to a complementary sequence. Accordingly, pluralities of ETMsare preferred, with at least about 2 ETMs per recruitment linker beingpreferred, and at least about 10 being particularly preferred, and atleast about 20 to 50 being especially preferred. In some instances, verylarge numbers of ETMs (50 to 1000) can be used.

Thus, solution binding ligands, or label probes, with covalentlyattached ETMs are provided. The method of attachment of the ETM to thesolution binding ligand will vary depending on the mode of detection(i.e. mechanism-1 or -2 systems) and the composition of the solutionbinding ligand. As is more fully outlined below, in mechanism-2 systems,the portion of the solution binding ligand (or label probe) thatcomprises the ETM is referred to as a “recruitment linker” and cancomprise either nucleic acid or non-nucleic acid. For mechanism-1systems, the recruitment linker must be nucleic acid.

Thus, as will be appreciated by those in the art, there are a variety ofconfigurations that can be used. In a preferred embodiment, therecruitment linker is nucleic acid (including analogs), and, attachmentof the ETMs can be via (1) a base; (2) the backbone, including theribose, the phosphate, or comparable structures in nucleic acid analogs;(3) nucleoside replacement, described below; or (4) metallocenepolymers, as described below. In a preferred embodiment, the recruitmentlinker is non-nucleic acid, and can be either a metallocene polymer oran alkyl-type polymer (including heteroalkyl, as is more fully describedbelow) containing ETM substitution groups.

In a preferred embodiment, the recruitment linker is a nucleic acid, andcomprises covalently attached ETMs. The ETMs may be attached tonucleosides within the nucleic acid in a variety of positions. Preferredembodiments include, but are not limited to, (1) attachment to the baseof the nucleoside, (2) attachment of the ETM as a base replacement, (3)attachment to the backbone of the nucleic acid, including either to aribose of the ribose-phosphate backbone or to a phosphate moiety, or toanalogous structures in nucleic acid analogs, and (4) attachment viametallocene polymers.

In addition, as is described below, when the recruitment linker isnucleic acid, it may be desirable to use secondary label probes, thathave a first portion that will hybridize to a portion of the primarylabel probes and a second portion comprising a recruitment linker as isdefined herein. This is similar to the use of an amplifier probe, exceptthat both the primary and the secondary label probes comprise ETMs.

In a preferred embodiment, the ETM is attached to the base of anucleoside as is generally outlined above for attachment of theconductive oligomer. Attachment can be to an internal nucleoside or aterminal nucleoside.

The covalent attachment to the base will depend in part on the ETMchosen, but in general is similar to the attachment of conductiveoligomers to bases, as outlined above. Attachment may generally be doneto any position of the base. In a preferred embodiment, the ETM is atransition metal complex, and thus attachment of a suitable metal ligandto the base leads to the covalent attachment of the ETM. Alternatively,similar types of linkages may be used for the attachment of organicETMs, as will be appreciated by those in the art.

In one embodiment, the C4 attached amino group of cytosine, the C6attached amino group of adenine, or the C2 attached amino group ofguanine may be used as a transition metal ligand.

Ligands containing aromatic groups can be attached via acetylenelinkages as is known in the art (see Comprehensive Organic Synthesis,Trost et al., Ed., Pergamon Press, Chapter 2.4: Coupling ReactionsBetween sp² and sp Carbon Centers, Sonogashira, pp 521-549, and pp950-953, hereby incorporated by reference). Structure 30 depicts arepresentative structure in the presence of the metal ion and any othernecessary ligands; Structure 30 depicts uridine, although as for all thestructures herein, any other base may also be used.

L_(a) is a ligand, which may include nitrogen, oxygen, sulfur orphosphorus donating ligands or organometallic ligands such asmetallocene ligands. Suitable L_(a) ligands include, but not limited to,phenanthroline, imidazole, bpy and terpy. L_(r) and M are as definedabove. Again, it will be appreciated by those in the art, a linker (“Z”)may be included between the nucleoside and the ETM.

Similarly, as for the conductive oligomers, the linkage may be doneusing a linker, which may utilize an amide linkage (see generally Telseret al., J. Am. Chem. Soc. 111:7221-7226 (1989); Telser et al., J. Am.Chem. Soc. 111:7226-7232 (1989), both of which are expresslyincorporated by reference). These structures are generally depictedbelow in Structure 31, which again uses uridine as the base, although asabove, the other bases may also be used:

In this embodiment, L is a ligand as defined above, with L, and M asdefined above as well. Preferably, L is amino, phen, byp and terpy.

In a preferred embodiment, the ETM attached to a nucleoside is ametallocene; i.e. the L and L_(r) of Structure 31 are both metalloceneligands, L_(m′) as described above. Structure 32 depicts a preferredembodiment wherein the metallocene is ferrocene, and the base isuridine, although other bases may be used:

Preliminary data suggest that Structure 32 may cyclize, with the secondacetylene carbon atom attacking the carbonyl oxygen, forming afuran-like structure. Preferred metallocenes include ferrocene,cobaltocene and osmiumocene.

In a preferred embodiment, the ETM is attached to a ribose at anyposition of the ribose-phosphate backbone of the nucleic acid, i.e.either the 5′ or 3′ terminus or any internal nucleoside. Ribose in thiscase can include ribose analogs. As is known in the art, nucleosidesthat are modified at either the 2′ or 3′ position of the ribose can bemade, with nitrogen, oxygen, sulfur and phosphorus-containingmodifications possible. Amino-modified and oxygen-modified ribose ispreferred. See generally PCT publication WO 95/15971, incorporatedherein by reference. These modification groups may be used as atransition metal ligand, or as a chemically functional moiety forattachment of other transition metal ligands and organometallic ligands,or organic electron donor moieties as will be appreciated by those inthe art. In this embodiment, a linker such as depicted herein for “Z”may be used as well, or a conductive oligomer between the ribose and theETM. Preferred embodiments utilize attachment at the 2′ or 3′ positionof the ribose, with the 2′ position being preferred. Thus for example,the conductive oligomers depicted in Structure 13, 14 and 15 may bereplaced by ETMs; alternatively, the ETMs may be added to the freeterminus of the conductive oligomer.

In a preferred embodiment, a metallocene serves as the ETM, and isattached via an amide bond as depicted below in Structure 33. Theexamples outline the synthesis of a preferred compound when themetallocene is ferrocene.

In a preferred embodiment, amine linkages are used, as is generallydepicted in Structure 34.

Z is a linker, as defined herein, with 1-16 atoms being preferred, and2-4 atoms being particularly preferred, and t is either one or zero.

In a preferred embodiment, oxo linkages are used, as is generallydepicted in Structure 35.

In Structure 35, Z is a linker, as defined herein, and t is either oneor zero. Preferred Z linkers include alkyl groups including heteroalkylgroups such as (CH₂)_(n) and (CH₂CH₂O)_(n), with n from 1 to 10 beingpreferred, and n=1 to 4 being especially preferred, and n=4 beingparticularly preferred.

Linkages utilizing other heteroatoms are also possible.

In a preferred embodiment, an ETM is attached to a phosphate at anyposition of the ribose-phosphate backbone of the nucleic acid. This maybe done in a variety of ways. In one embodiment, phosphodiester bondanalogs such as phosphoramide or phosphoramidite linkages may beincorporated into a nucleic acid, where the heteroatom (i.e. nitrogen)serves as a transition metal ligand (see PCT publication WO 95/15971,incorporated by reference). Alternatively, the conductive oligomersdepicted in Structures 23 and 24 may be replaced by ETMs. In a preferredembodiment, the composition has the structure shown in Structure 36,

In Structure 36, the ETM is attached via a phosphate linkage, generallythrough the use of a linker, Z. Preferred Z linkers include alkylgroups, including heteroalkyl groups such as (CH₂)_(n), (CH₂CH₂O)_(n),with n from 1 to 10 being preferred, and n=1 to 4 being especiallypreferred, and n=4 being particularly preferred.

In mechanism-2 systems, when the ETM is attached to the base or thebackbone of the nucleoside, it is possible to attach the ETMs via“dendrimer” structures, as is more fully outlined below. As is generallydepicted in FIG. 8, alkyl-based linkers can be used to create multiplebranching structures comprising one or more ETMs at the terminus of eachbranch. Generally, this is done by creating branch points containingmultiple hydroxy groups, which optionally can then be used to addadditional branch points. The terminal hydroxy groups can then be usedin phosphoramidite reactions to add ETMs, as is generally done below forthe nucleoside replacement and metallocene polymer reactions.

In a preferred embodiment, an ETM such as a metallocene is used as a“nucleoside replacement”, serving as an ETM. For example, the distancebetween the two cyclopentadiene rings of ferrocene is similar to theorthongonal distance between two bases in a double stranded nucleicacid. Other metallocenes in addition to ferrocene may be used, forexample, air stable metallocenes such as those containing cobalt orruthenium. Thus, metallocene moieties may be incorporated into thebackbone of a nucleic acid, as is generally depicted in Structure 37(nucleic acid with a ribose-phosphate backbone) and Structure 38(peptide nucleic acid backbone). Structures 37 and 38 depict ferrocene,although as will be appreciated by those in the art, other metallocenesmay be used as well. In general, air stable metallocenes are preferred,including metallocenes utilizing ruthenium and cobalt as the metal.

In Structure 37, Z is a linker as defined above, with generally short,alkyl groups, including heteroatoms such as oxygen being preferred.Generally, what is important is the length of the linker, such thatminimal perturbations of a double stranded nucleic acid is effected, asis more fully described below. Thus, methylene, ethylene, ethyleneglycols, propylene and butylene are all preferred, with ethylene andethylene glycol being particularly preferred. In addition, each Z linkermay be the same or different. Structure 37 depicts a ribose-phosphatebackbone, although as will be appreciated by those in the art, nucleicacid analogs may also be used, including ribose analogs and phosphatebond analogs.

In Structure 38, preferred Z groups are as listed above, and again, eachZ linker can be the same or different. As above, other nucleic acidanalogs may be used as well.

In addition, although the structures and discussion above depictsmetallocenes, and particularly ferrocene, this same general idea can beused to add ETMs in addition to metallocenes, as nucleoside replacementsor in polymer embodiments, described below. Thus, for example, when theETM is a transition metal complex other than a metallocene, comprisingone, two or three (or more) ligands, the ligands can be functionalizedas depicted for the ferrocene to allow the addition of phosphoramiditegroups. Particularly preferred in this embodiment are complexescomprising at least two ring (for example, aryl and substituted aryl)ligands, where each of the ligands comprises functional groups forattachment via phosphoramidite chemistry. As will be appreciated bythose in the art, this type of reaction, creating polymers of ETMseither as a portion of the backbone of the nucleic acid or as “sidegroups” of the nucleic acids, to allow amplification of the signalsgenerated herein, can be done with virtually any ETM that can befunctionalized to contain the correct chemical groups.

Thus, by inserting a metallocene such as ferrocene (or other ETM) intothe backbone of a nucleic acid, nucleic acid analogs are made; that is,the invention provides nucleic acids having a backbone comprising atleast one metallocene. This is distinguished from nucleic acids havingmetallocenes attached to the backbone, i.e. via a ribose, a phosphate,etc. That is, two nucleic acids each made up of a traditional nucleicacid or analog (nucleic acids in this case including a singlenucleoside), may be covalently attached to each other via a metallocene.Viewed differently, a metallocene derivative or substituted metalloceneis provided, wherein each of the two aromatic rings of the metallocenehas a nucleic acid substitutent group.

In addition, as is more fully outlined below, it is possible toincorporate more than one metallocene into the backbone, either withnucleotides in between and/or with adjacent metallocenes. When adjacentmetallocenes are added to the backbone, this is similar to the processdescribed below as “metallocene polymers”: that is, there are areas ofmetallocene polymers within the backbone.

In addition to the nucleic acid substitutent groups, it is alsodesirable in some instances to add additional substituent groups to oneor both of the aromatic rings of the metallocene (or ETM). For example,as these nucleoside replacements are generally part of probe sequencesto be hybridized with a substantially complementary nucleic acid, forexample a target sequence or another probe sequence, it is possible toadd substitutent groups to the metallocene rings to facilitate hydrogenbonding to the base or bases on the opposite strand. These may be addedto any position on the metallocene rings. Suitable substitutent groupsinclude, but are not limited to, amide groups, amine groups, carboxylicacids, and alcohols, including substituted alcohols. In addition, thesesubstitutent groups can be attached via linkers as well, although ingeneral this is not preferred.

In addition, substituent groups on an ETM, particularly metallocenessuch as ferrocene, may be added to alter the redox properties of theETM. Thus, for example, in some embodiments, as is more fully describedbelow, it may be desirable to have different ETMs attached in differentways (i.e. base or ribose attachment), on different probes, or fordifferent purposes (for example, calibration or as an internalstandard). Thus, the addition of substituent groups on the metallocenemay allow two different ETMs to be distinguished.

In order to generate these metallocene-backbone nucleic acid analogs,the intermediate components are also provided. Thus, in a preferredembodiment, the invention provides phosphoramidite metallocenes, asgenerally depicted in Structure 39:

In Structure 39, PG is a protecting group, generally suitable for use innucleic acid synthesis, with DMT, MMT and TMT all being preferred. Thearomatic rings can either be the rings of the metallocene, or aromaticrings of ligands for transition metal complexes or other organic ETMs.The aromatic rings may be the same or different, and may be substitutedas discussed herein.

Structure 40 depicts the ferrocene derivative:

These phosphoramidite analogs can be added to standard oligonucleotidesyntheses as is known in the art.

Structure 41 depicts the ferrocene peptide nucleic acid (PNA) monomer,that can be added to PNA synthesis as is known in the art:

In Structure 41, the PG protecting group is suitable for use in peptidenucleic acid synthesis, with MMT, boc and Fmoc being preferred.

These same intermediate compounds can be used to form ETM or metallocenepolymers, which are added to the nucleic acids, rather than as backbonereplacements, as is more fully described below.

In a preferred embodiment, particularly for use in mechanism-2 systems,the ETMs are attached as polymers, for example as metallocene polymers,in a “branched” configuration similar to the “branched DNA” embodimentsherein and as outlined in U.S. Pat. No. 5,124,246, using modifiedfunctionalized nucleotides. The general idea is as follows. A modifiedphosphoramidite nucleotide is generated that can ultimately contain afree hydroxy group that can be used in the attachment of phosphoramidite

ETMs such as metallocenes. This free hydroxy group could be on the baseor the backbone, such as the ribose or the phosphate (although as willbe appreciated by those in the art, nucleic acid analogs containingother structures can also be used). The modified nucleotide isincorporated into a nucleic acid, and any hydroxy protecting groups areremoved, thus leaving the free hydroxyl. Upon the addition of aphosphoramidite ETM such as a metallocene, as described above instructures 39 and 40, ETMs, such as metallocene ETMs, are added.Additional phosphoramidite ETMs such as metallocenes can be added, toform “ETM polymers”, including “metallocene polymers” as depicted inFIG. 9 with ferrocene. In addition, in some embodiments, it is desirableto increase the solubility of the polymers by adding a “capping” groupto the terminal ETM in the polymer, for example a final phosphate groupto the metallocene. Other suitable solubility enhancing “capping” groupswill be appreciated by those in the art. It should be noted that thesesolubility enhancing groups can be added to the polymers in otherplaces, including to the ligand rings, for example on the metallocenesas discussed herein.

In this embodiment, the 2′ position of a ribose of a phosphoramiditenucleotide is first functionalized to contain a protected hydroxy group,in this case via an oxo-linkage, although any number of linkers can beused, as is generally described herein for Z linkers. The protectedmodified nucleotide is then incorporated via standard phosphoramiditechemistry into a growing nucleic acid. The protecting group is removed,and the free hydroxy group is used, again using standard phosphoramiditechemistry to add a phosphoramidite metallocene such as ferrocene. Asimilar reaction is possible for nucleic acid analogs. For example,using peptide nucleic acids and the metallocene monomer shown inStructure 41, peptide nucleic acid structures containing metallocenepolymers could be generated.

Thus, the present invention provides recruitment linkers of nucleicacids comprising “branches” of metallocene polymers. Preferredembodiments also utilize metallocene polymers from one to about 50metallocenes in length, with from about 5 to about 20 being preferredand from about 5 to about 10 being especially preferred.

In addition, when the recruitment linker is nucleic acid, anycombination of ETM attachments may be done. In general, as outlinedherein, when mechanism-1 systems are used, clusters of nucleosidescontaining ETMs can decrease the Tm of hybridization of the probe to itstarget sequence; thus in general, for mechanism-1 systems, the ETMs arespaced out over the length of the sequence, or only small numbers ofthem are used.

In mechanism-1 systems, non-covalently attached ETMs may be used. In oneembodiment, the ETM is a hybridization indicator. Hybridizationindicators serve as an ETM that will preferentially associate withdouble stranded nucleic acid is added, usually reversibly, similar tothe method of Millan et al., Anal. Chem. 65:231.7-2323 (1993); Millan etal., Anal. Chem. 662943-2948 (1994), both of which are hereby expresslyincorporated by reference. In this embodiment, increases in the localconcentration of ETMs, due to the association of the ETM hybridizationindicator with double stranded nucleic acid at the surface, can bemonitored using the monolayers comprising the conductive oligomers.Hybridization indicators include intercalators and minor and/or majorgroove binding moieties. In a preferred embodiment, intercalators may beused; since intercalation generally only occurs in the presence ofdouble stranded nucleic acid, only in the presence of double strandednucleic acid will the ETMs concentrate. Intercalating transition metalcomplex ETMs are known in the art. Similarly, major or minor groovebinding moieties, such as methylene blue, may also be used in thisembodiment.

Similarly, the systems of the invention may utilize non-covalentlyattached ETMs, as is generally described in Napier et al., Bioconj.Chem., 8:906 (1997), hereby expressly incorporated by reference. In thisembodiment, changes in the redox state of certain molecules as a resultof the presence of DNA (i.e. guanine oxidation by ruthenium complexes)can be detected using the SAMs comprising conductive oligomers as well.

In a preferred embodiment, the recruitment linker is not nucleic acid,and instead may be any sort of linker or polymer. As will be appreciatedby those in the art, generally any linker or polymer that can bemodified to contain ETMs can be used. In general, the polymers orlinkers should be reasonably soluble and contain suitable functionalgroups for the addition of ETMs.

As used herein, a “recruitment polymer” comprises at least two or threesubunits, which are covalently attached. At least some portion of themonomeric subunits contain functional groups for the covalent attachmentof ETMs. In some embodiments coupling moieties are used to covalentlylink the subunits with the ETMs. Preferred functional groups forattachment are amino groups, carboxy groups, oxo groups and thiolgroups, with amino groups being particularly preferred. As will beappreciated by those in the art, a wide variety of recruitment polymersare possible.

Suitable linkers include, but are not limited to, alkyl linkers(including heteroalkyl (including (poly)ethylene glycol-typestructures), substituted alkyl, aryalkyl linkers, etc. As above for thepolymers, the linkers will comprise one or more functional groups forthe attachment of ETMs, which will be done as will be appreciated bythose in the art, for example through the use homo- orheterobifunctional linkers as are well known (see 1994 Pierce ChemicalCompany catalog, technical section on cross-linkers, pages 155-200,incorporated herein by reference).

Suitable recruitment polymers include, but are not limited to,functionalized styrenes, such as amino styrene, functionalized dextrans,and polyamino acids. Preferred polymers are polyamino acids (bothpoly-D-amino acids and poly-L-amino acids), such as polylysine, andpolymers containing lysine and other amino acids being particularlypreferred. As outlined above, in some embodiments, charged recruitmentlinkers are preferred, for example when non-charged target analytes areto be detected. Other suitable polyamino acids are polyglutamic acid,polyaspartic acid, co-polymers of lysine and glutamic or aspartic acid,co-polymers of lysine with alanine, tyrosine, phenylalanine, serine,tryptophan, and/or proline.

In a preferred embodiment, the recruitment linker comprises ametallocene polymer, as is described above.

The attachment of the recruitment linkers to the first portion of thelabel probe, i.e. the portion that binds either directly or indirectlyto the target analyte, will depend on the composition of the recruitmentlinker, as will be appreciated by those in the art. When the recruitmentlinker is nucleic acid, it is generally formed during the synthesis ofthe first portion of the label probe, with incorporation of nucleosidescontaining ETMs as required. Alternatively, the first portion of thelabel probe and the recruitment linker may be made separately, and thenattached. For example, there may be an overlapping section ofcomplementarity, forming a section of double stranded nucleic acid thatcan then be chemically crosslinked, for example by using psoralen as isknown in the art.

When non-nucleic acid recruitment linkers are used, attachment of thelinker/polymer of the recruitment linker will be done generally usingstandard chemical techniques, such as will be appreciated by those inthe art. For example, when alkyl-based linkers are used, attachment canbe similar to the attachment of insulators to nucleic acids.

In addition, it is possible to have recruitment linkers that aremixtures of nucleic acids and non-nucleic acids, either in a linear form(i.e. nucleic acid segments linked together with alkyl linkers) or inbranched forms (nucleic acids with alkyl “branches” that may containETMs and may be additionally branched).

In a preferred embodiment, for example when the target analyte is anucleic acid, it is the target sequence itself that carries the ETMs,rather than the recruitment linker of a label probe. For example, as ismore fully described below, it is possible to enzymatically addtriphosphate nucleotides comprising the ETMs of the invention to agrowing nucleic acid, for example during a polymerase chain reaction(PCR). As will be recognized by those in the art, while several enzymeshave been shown to generally tolerate modified nucleotides, some of themodified nucleotides of the invention, for example the “nucleosidereplacement” embodiments and putatively some of the phosphateattachments, may or may not be recognized by the enzymes to allowincorporation into a growing nucleic acid. Therefore, preferredattachments in this embodiment are to the base or ribose of thenucleotide.

Thus, for example, PCR amplification of a target sequence, as is wellknown in the art, will result in target sequences comprising ETMs,generally randomly incorporated into the sequence. The system of theinvention can then be configured to allow detection using these ETMs.

Alternatively, as outlined more fully below, it is possible toenzymatically add nucleotides comprising ETMs to the terminus of anucleic acid, for example a target nucleic acid. In this embodiment, aneffective “recruitment linker” is added to the terminus of the targetsequence, that can then be used for detection. Thus the inventionprovides compositions utilizing electrodes comprising monolayers ofconductive oligomers and capture probes, and target sequences thatcomprises a first portion that is capable of hybridizing to a componentof an assay complex, and a second portion that does not hybridize to acomponent of an assay complex and comprises at least one covalentlyattached electron transfer moiety. Similarly, methods utilizing thesecompositions are also provided.

It is also possible to have ETMs connected to probe sequences, i.e.sequences designed to hybridize to complementary sequences, i.e. inmechanism-1 sequences, although this may also be used in mechanism-2systems. Thus, ETMs may be added to non-recruitment linkers as well. Forexample, there may be ETMs added to sections of label probes that dohybridize to components of the assay complex, for example the firstportion, or to the target sequence as outlined above. These ETMs may beused for electron transfer detection in some embodiments, or they maynot, depending on the location and system. For example, in someembodiments, when for example the target sequence containing randomlyincorporated ETMs is hybridized directly to the capture probe, there maybe ETMs in the portion hybridizing to the capture probe. If the captureprobe is attached to the electrode using a conductive oligomer, theseETMs can be used to detect electron transfer as has been previouslydescribed. Alternatively, these ETMs may not be specifically detected.

Similarly, in some embodiments, when the recruitment linker is nucleicacid, it may be desirable in some instances to have some or all of therecruitment linker be double stranded, for example in the mechanism-2systems. In one embodiment, there may be a second recruitment linker,substantially complementary to the first recruitment linker, that canhybridize to the first recruitment linker. In a preferred embodiment,the first recruitment linker comprises the covalently attached ETMs. Inan alternative embodiment, the second recruitment linker contains theETMs, and the first recruitment linker does not, and the ETMs arerecruited to the surface by hybridization of the second recruitmentlinker to the first. In yet another embodiment, both the first andsecond recruitment linkers comprise ETMs. It should be noted, asdiscussed above, that nucleic acids comprising a large number of ETMsmay not hybridize as well, i.e. the T_(m) may be decreased, depending onthe site of attachment and the characteristics of the ETM. Thus, ingeneral, when multiple ETMs are used on hybridizing strands, i.e. inmechanism-1 systems, generally there are less than about 5, with lessthan about 3 being preferred, or alternatively the ETMs should be spacedsufficiently far apart that the intervening nucleotides can sufficientlyhybridize to allow good kinetics.

Thus, the present invention provides compositions comprising detectionelectrodes comprising monolayers comprising conductive oligomers,generally including capture probes, and either target sequences or labelprobes comprising recruitment linkers containing ETMs. In a preferredembodiment, the compositions of the invention are used to detect targetanalytes in a sample. In a preferred embodiment, the target analyte is anucleic acid, and target sequences are detected.

As will be appreciated by those in the art, the systems of the inventionmay take on a large number of different configurations. In general,there are three types of systems that can be used: (1) systems in whichthe target sequence itself is labeled with ETMs; this is generallyuseful for nucleic acid systems); (2) systems in which label probesdirectly bind to the target analytes; and (3) systems in which labelprobes are indirectly bound to the target sequences, for example throughthe use of amplifier probes

In a preferred embodiment, the target sequence itself contains the ETMs.As discussed above, this may be done using target sequences that haveETMs incorporated at any number of positions, as outlined above. In thisembodiment, as for the others of the system, the 3′-5′ orientation ofthe probes and targets is chosen to get the ETM-containing structures(i.e. recruitment linkers or target sequences) as close to the surfaceof the monolayer as possible, and in the correct orientation. This maybe done using attachment via insulators or conductive oligomers as isgenerally shown in the Figures. In addition, as will be appreciated bythose in the art, multiple capture probes can be utilized, for examplein a configuration, wherein the 5′-3′ orientation of the capture probesis different, or where “loops” of target form when multiples of captureprobes are used.

In a preferred embodiment, the label probes directly hybridize to thetarget sequences. In these embodiments, the target sequence ispreferably, but not required to be, immobilized on the surface usingcapture probes, including capture extender probes. Label probes are thenused to bring the ETMs into proximity of the surface of the monolayercomprising conductive oligomers. In a preferred embodiment, multiplelabel probes are used; that is, label probes are designed such that theportion that hybridizes to the target sequence can be different for anumber of different label probes, such that amplification of the signaloccurs, since multiple label probes can bind for every target sequence.Thus, as depicted in the figures, n is an integer of at least one.Depending on the sensitivity desired, the length of the target sequence,the number of ETMs per label probe, etc., preferred ranges of n are from1 to 50, with from about 1 to about 20 being particularly preferred, andfrom about 2 to about 5 being especially preferred. In addition, if“generic” label probes are desired, label extender probes can be used asgenerally described below for use with amplifier probes.

As above, generally in this embodiment the configuration of the systemand the label probes are designed to recruit the ETMs as close aspossible to the monolayer surface.

In a preferred embodiment, the label probes are hybridized to the targetsequence indirectly. That is, the present invention finds use in novelcombinations of signal amplification technologies and electron transferdetection on electrodes, which may be particularly useful in sandwichhybridization assays, as generally depicted in the Figures for nucleicacid embodiments; similar systems can be developed for non-nucleic acidtarget analytes. In these embodiments, the amplifier probes of theinvention are bound to the target sequence in a sample either directlyor indirectly. Since the amplifier probes preferably contain arelatively large number of amplification sequences that are availablefor binding of label probes, the detectable signal is significantlyincreased, and allows the detection limits of the target to besignificantly improved. These label and amplifier probes, and thedetection methods described herein, may be used in essentially any knownnucleic acid hybridization formats, such as those in which the target isbound directly to a solid phase or in sandwich hybridization assays inwhich the target is bound to one or more nucleic acids that are in turnbound to the solid phase.

In general, these embodiments may be described as follows withparticular reference to nucleic acids. An amplifier probe is hybridizedto the target sequence, either directly or through the use of a labelextender probe, which serves to allow “generic” amplifier probes to bemade. The target sequence is preferably, but not required to be,immobilized on the electrode using capture probes. Preferably, theamplifier probe contains a multiplicity of amplification sequences,although in some embodiments, as described below, the amplifier probemay contain only a single amplification sequencer. The amplifier probemay take on a number of different forms; either a branched conformation,a dendrimer conformation, or a linear “string” of amplificationsequences. These amplification sequences are used to form hybridizationcomplexes with label probes, and the ETMs can be detected using theelectrode.

The reactions outlined herein may be accomplished in a variety of ways,as will be appreciated by those in the art. Components of the reactionmay be added simultaneously, or sequentially, in any order, withpreferred embodiments outlined below. In addition, the reaction mayinclude a variety of other reagents may be included in the assays. Theseinclude reagents like salts, buffers, neutral proteins, e.g. albumin,detergents, etc which may be used to facilitate optimal hybridizationand detection, and/or reduce non-specific or background interactions.Also reagents that otherwise improve the efficiency of the assay, suchas protease inhibitors, nuclease inhibitors, anti-microbial agents,etc., may be used, depending on the sample preparation methods andpurity of the target.

Generally, the methods are as follows. In a preferred embodiment, thetarget is moved into the detection module. In general, two methods maybe employed; the assay complexes as described below are formed first(i.e. all the soluble components are added together, eithersimultaneously or sequentially, including capture extender probes, labelprobes, amplification probes, label extender probes, etc.), “upstream”of the detection module, and then the complex is added to the surfacefor subsequent binding to a detection electrode. Alternatively, thetarget may be added where it binds the capture binding ligand and thenadditional components are added. The latter is described in detailbelow, but either procedure may be followed. Similarly, some componentsmay be added, electrophoresed, and other components added; for example,the target analyte may be combined with any capture extender probes andthen transported, etc. In addition, as outlined herein, “washing” stepsmay be done using the introduction of buffer into the detection module,wherein excess reagents (non-bound analytes, excess probes, etc.) can bedriven from the surface.

The sample is introduced to the electrode in the detection module, andthen immobilized or attached to the detection electrode. In oneembodiment, this is done by forming an attachment complex (frequentlyreferred to herein as a hybridization complex when nucleic acidcomponents are used) between a capture probe and a portion of the targetanalyte. A preferred embodiment utilizes capture extender bindingligands (also called capture extender probes herein); in thisembodiment, an attachment complex is formed between a portion of thetarget sequence and a first portion of a capture extender probe, and anadditional attachment complex between a second portion of the captureextender probe and a portion of the capture probe. Additional preferredembodiments utilize additional capture probes, thus forming anattachment complex between a portion of the target sequence and a firstportion of a second capture extender probe, and an attachment complexbetween a second portion of the second capture extender probe and asecond portion of the capture probe.

Alternatively, the attachment of the target sequence to the electrode isdone simultaneously with the other reactions.

The method proceeds with the introduction of amplifier probes, ifutilized. In a preferred embodiment, the amplifier probe comprises afirst probe sequence that is substantially complementary to a portion ofthe target sequence, and at least one amplification sequence.

In one embodiment, the first probe sequence of the amplifier probe ishybridized to the target sequence, and any unhybridized amplifier probeis removed. This will generally be done as is known in the art, anddepends on the type of assay. When the target sequence is immobilized ona surface such as an electrode, the removal of excess reagents generallyis done via one or more washing steps, as will be appreciated by thosein the art. In this embodiment, the target may be immobilized on anysolid support. When the target sequence is not immobilized on a surface,the removal of excess reagents such as the probes of the invention maybe done by flowing the sample past a solid support that containcomplementary sequences to the probes, such that the excess probes bindto the solid support.

The reaction mixture is then subjected to conditions (temperature, highsalt, changes in pH, etc.) under which the amplifier probe disassociatesfrom the target sequence, and the amplifier probe is collected. Theamplifier probe may then be added to an electrode comprising captureprobes for the amplifier probes, label probes added, and detection isachieved.

In a preferred embodiment, a larger pool of probe is generated by addingmore amplifier probe to the target sequence and thehybridization/disassociation reactions are repeated, to generate alarger pool of amplifier probe. This pool of amplifier probe is thenadded to an electrode comprising amplifier capture probes, label probesadded, and detection proceeds.

In this embodiment, it is preferred that the target sequence beimmobilized on a solid support, including an electrode, using themethods described herein; although as will be appreciated by those inthe art, alternate solid support attachment technologies may be used,such as attachment to glass, polymers, etc. It is possible to do thereaction on one solid support and then add the pooled amplifier probe toan electrode for detection.

In a preferred embodiment, the amplifier probe comprises a multiplicityof amplification sequences.

In one embodiment, the first probe sequence of the amplifier probe ishybridized to the target sequence, and any unhybridized amplifier probeis removed. Again, preferred embodiments utilize immobilized targetsequences, wherein the target sequences are immobilized by hybridizationwith capture probes that are attached to the electrode, or hybridizationto capture extender probes that in turn hybridize with immobilizedcapture probes as is described herein. Generally, in these embodiments,the capture probes and the detection probes are immobilized on theelectrode, generally at the same “address”.

In a preferred embodiment, the first probe sequence of the amplifierprobe is hybridized to a first portion of at least one label extenderprobe, and a second portion of the label extender probe is hybridized toa portion of the target sequence. Other preferred embodiments utilizemore than one label extender probe.

In a preferred embodiment, the amplification sequences of the amplifierprobe are used directly for detection, by hybridizing at least one labelprobe sequence.

The invention thus provides assay complexes that minimally comprise atarget sequence and a label probe. “Assay complex” herein is meant thecollection of attachment or hybridization complexes comprising analytes,including binding ligands and targets, that allows detection. Thecomposition of the assay complex depends on the use of the differentprobe component outlined herein. The assay complexes may include thetarget sequence, label probes, capture extender probes, label extenderprobes, and amplifier probes, as outlined herein, depending on theconfiguration used.

The assays are generally run under stringency conditions which allowsformation of the label probe attachment complex only in the presence oftarget. Stringency can be controlled by altering a step parameter thatis a thermodynamic variable, including, but not limited to, temperature,formamide concentration, salt concentration, chaotropic saltconcentration pH, organic solvent concentration, etc. Stringency mayalso include the use of an electrophoretic step to drive non-specific(i.e. low stringency) materials away from the detection electrode.

These parameters may also be used to control non-specific binding, as isgenerally outlined in U.S. Pat. No. 5,681,697. Thus it may be desirableto perform certain steps at higher stringency conditions; for example,when an initial hybridization step is done between the target sequenceand the label extender and capture extender probes. Running this step atconditions which favor specific binding can allow the reduction ofnon-specific binding.

In a preferred nucleic acid embodiment, when all of the componentsoutlined herein are used, a preferred method is as follows.Single-stranded target sequence is incubated under hybridizationconditions with the capture extender probes and the label extenderprobes. A preferred embodiment does this reaction in the presence of theelectrode with immobilized capture probes, although this may also bedone in two steps, with the initial incubation and the subsequentaddition to the electrode. Excess reagents are washed off, and amplifierprobes are then added. If preamplifier probes are used, they may beadded either prior to the amplifier probes or simultaneously with theamplifier probes. Excess reagents are washed off, and label probes arethen added. Excess reagents are washed off, and detection proceeds asoutlined below.

In one embodiment, a number of capture probes (or capture probes andcapture extender probes) that are each substantially complementary to adifferent portion of the target sequence are used.

Again, as outlined herein, when amplifier probes are used, the system isgenerally configured such that upon label probe binding, the recruitmentlinkers comprising the ETMs are placed in proximity either to themonolayer surface containing conductive oligomers (mechanism-2) or inproximity to detection probes. Thus for example, for mechanism-2systems, when the ETMs are attached via “dendrimer” type structures asoutlined herein, the length of the linkers from the nucleic acid pointof attachment to the ETMs may vary, particularly with the length of thecapture probe when capture extender probes are used. That is, longercapture probes, with capture extenders, can result in the targetsequences being “held” further away from the surface than for shortercapture probes. Adding extra linking sequences between the probe nucleicacid and the ETMs can result in the ETMs being spatially closer to thesurface, giving better results. Similarly, for mechanism-1 systems, thelength of the recruitment linker, the length of the detection probe, andtheir distance, may be optimized.

In addition, if desirable, nucleic acids utilized in the invention mayalso be ligated together prior to detection, if applicable, by usingstandard molecular biology techniques such as the use of a ligase.Similarly, if desirable for stability, cross-linking agents may be addedto hold the structures stable.

As will be appreciated by those in the art, while described for nucleicacids, the systems outlined herein can be used for other target analytesas well.

The compositions of the invention are generally synthesized as outlinedherein and in U.S. Ser. Nos. 08/743,798, 08/873,978, 08/911,085,08/911,085, and PCT US97/20014, all of which are expressly incorporatedby reference, generally utilizing techniques well known in the art. Aswill be appreciated by those in the art, many of the techniques outlinedbelow are directed to nucleic acids containing a ribose-phosphatebackbone. However, as outlined above, many alternate nucleic acidanalogs may be utilized, some of which may not contain either ribose orphosphate in the backbone. In these embodiments, for attachment atpositions other than the base, attachment is done as will be appreciatedby those in the art, depending on the backbone. Thus, for example,attachment can be made at the carbon atoms of the PNA backbone, as isdescribed below, or at either terminus of the PNA.

The compositions may be made in several ways. A preferred method firstsynthesizes a conductive oligomer attached to a nucleoside, withaddition of additional nucleosides to form the capture probe followed byattachment to the electrode. Alternatively, the whole capture probe maybe made and then the completed conductive oligomer added, followed byattachment to the electrode. Alternatively, a monolayer of conductiveoligomer (some of which have functional groups for attachment of captureprobes) is attached to the electrode first, followed by attachment ofthe capture probe. The latter two methods may be preferred whenconductive oligomers are used which are not stable in the solvents andunder the conditions used in traditional nucleic acid synthesis.

In a preferred embodiment, the detection module compositions of theinvention are made by first forming the conductive oligomer covalentlyattached to the nucleoside, followed by the addition of additionalnucleosides to form a capture probe nucleic acid, with the last stepcomprising the addition of the conductive oligomer to the electrode.

The attachment of the conductive oligomer to the nucleoside may be donein several ways. In a preferred embodiment, all or part of theconductive oligomer is synthesized first (generally with a functionalgroup on the end for attachment to the electrode), which is thenattached to the nucleoside. Additional nucleosides are then added asrequired, with the last step generally being attachment to theelectrode. Alternatively, oligomer units are added one at a time to thenucleoside, with addition of additional nucleosides and attachment tothe electrode. A number of representative syntheses are shown in theFigures of PCT US97/20014.

The conductive oligomer is then attached to a nucleoside that maycontain one (or more) of the oligomer units, attached as depictedherein.

In a preferred embodiment, attachment is to a ribose of theribose-phosphate backbone, including amide and amine linkages. In apreferred embodiment, there is at least a methylene group or other shortaliphatic alkyl groups (as a Z group) between the nitrogen attached tothe ribose and the aromatic ring of the conductive oligomer.

Alternatively, attachment is via a phosphate of the ribose-phosphatebackbone, as generally outlined in PCT US97/20014.

In a preferred embodiment, attachment is via the base. In a preferredembodiment, protecting groups may be added to the base prior to additionof the conductive oligomers, as is generally known in the art. Inaddition, the palladium cross-coupling reactions may be altered toprevent dimerization problems; i.e. two conductive oligomers dimerizing,rather than coupling to the base.

Alternatively, attachment to the base may be done by making thenucleoside with one unit of the oligomer, followed by the addition ofothers.

Once the modified nucleosides are prepared, protected and activated,prior to attachment to the electrode, they may be incorporated into agrowing oligonucleotide by standard synthetic techniques (Gait,Oligonucleotide Synthesis: A Practical Approach, IRL Press, Oxford, UK1984; Eckstein) in several ways.

In one embodiment, one or more modified nucleosides are converted to thetriphosphate form and incorporated into a growing oligonucleotide chainby using standard molecular biology techniques suct as with the use ofthe enzyme DNA polymerase. I, T4 DNA polymerase, T7 DNA polymerase, TaqDNA polymerase, reverse transcriptase, and RNA polymerases. For theincorporation of a 3′ modified nucleoside to a nucleic acid, terminaldeoxynucleotidyltransferase may be used. (Ratliff, Terminaldeoxynucleotidyltransferase. In The Enzymes, Vol 14A. P. D. Boyer ed. pp105-118. Academic Press, San Diego, Calif. 1981). Thus, the presentinvention provides deoxyribonucleoside triphosphates comprising acovalently attached ETM. Preferred embodiments utilize ETM attachment tothe base or the backbone, such as the ribose (preferably in the 2′position), as is generally depicted below in Structures 42 and 43:

Thus, in some embodiments, it may be possible to generate the nucleicacids comprising ETMs in situ. For example, a target sequence canhybridize to a capture probe (for example on the surface) in such a waythat the terminus of the target sequence is exposed, i.e. unhybridized.The addition of enzyme and triphosphate nucleotides labelled with ETMsallows the in situ creation of the label. Similarly, using labelednucleotides recognized by polymerases can allow simultaneous PCR anddetection; that is, the target sequences are generated in situ.

In a preferred embodiment, the modified nucleoside is converted to thephosphoramidite or Hphosphonate form, which are then used in solid-phaseor solution syntheses of oligonucleotides. In this way the modifiednucleoside, either for attachment at the ribose (i.e. amino- orthiol-modified nucleosides) or the base, is incorporated into theoligonucleotide at either an internal position or the 5′ terminus. Thisis generally done in one of two ways. First, the 5′ position of theribose is protected with 4′,4-dimethoxytrityl (DMT) followed by reactionwith either 2-cyanoethoxy-bisdiisopropylaminophosphine in the presenceof diisopropylammonium tetrazolide, or by reaction withchlorodiisopropylamino 2′-cyanoethyoxyphosphine, to give thephosphoramidite as is known in the art although other techniques may beused as will be appreciated by those in the art. See Gait, supra;Caruthers, Science 230:281 (1985), both of which are expresslyincorporated herein by reference.

For attachment of a group to the 3′ terminus, a preferred methodutilizes the attachment of the modified nucleoside (or the nucleosidereplacement) to controlled pore glass (CPG) or other oligomericsupports. In this embodiment, the modified nucleoside is protected atthe 5′ end with DMT, and then reacted with succinic anhydride withactivation. The resulting succinyl compound is attached to CPG or otheroligomeric supports as is known in the art. Further phosphoramiditenucleosides are added, either modified or not, to the 5′ end afterdeprotection. Thus, the present invention provides conductive oligomersor insulators covalently attached to nucleosides attached to solidoligomeric supports such as CPG, and phosphoramidite derivatives of thenucleosides of the invention.

The invention further provides methods of making label probes withrecruitment linkers comprising ETMs. These synthetic reactions willdepend on the character of the recruitment linker and the method ofattachment of the ETM, as will be appreciated by those in the art. Fornucleic acid recruitment linkers, the label probes are generally made asoutlined herein with the incorporation of ETMs at one or more positions.When a transition metal complex is used as the ETM, synthesis may occurin several ways. In a preferred embodiment, the ligand(s) are added to anucleoside, followed by the transition metal ion, and then thenucleoside with the transition metal complex attached is added to anoligonucleotide, i.e. by addition to the nucleic acid synthesizer.Alternatively, the ligand(s) may be attached, followed by incorporationinto a growing oligonucleotide chain, followed by the addition of themetal ion.

In a preferred embodiment, ETMs are attached to a ribose of theribose-phosphate backbone. This is generally done as is outlined hereinfor conductive oligomers, as described herein, and in PCT publication WO95/15971, using amino-modified or oxo-modified nucleosides, at eitherthe 2′ or 3′ position of the ribose. The amino group may then be usedeither as a ligand, for example as a transition metal ligand forattachment of the metal ion, or as a chemically functional group thatcan be used for attachment of other ligands or organic ETMs, for examplevia amide linkages, as will be appreciated by those in the art. Forexample, the examples describe the synthesis of nucleosides with avariety of ETMs attached via the ribose.

In a preferred embodiment, ETMs are attached to a phosphate of theribose-phosphate backbone. As outlined herein, this may be done usingphosphodiester analogs such as phosphoramidite bonds, see generally PCTpublication WO 95115971, or can be done in a similar manner to thatdescribed in PCT US97/20014, where the conductive oligomer is replacedby a transition metal ligand or complex or an organic ETM.

Attachment to alternate backbones, for example peptide nucleic acids oralternate phosphate linkages will be done as will be appreciated bythose in the art.

In a preferred embodiment, ETMs are attached to a base of thenucleoside. This may be done in a variety of ways. In one embodiment,amino groups of the base, either naturally occurring or added as isdescribed herein (see the figures, for example), are used either asligands for transition metal complexes or as a chemically functionalgroup that can be used to add other ligands, for example via an amidelinkage, or organic ETMs. This is done as will be appreciated by thosein the art. Alternatively, nucleosides containing halogen atoms attachedto the heterocyclic ring are commercially availabler. Acetylene linkedligands may be added using the halogenated bases, as is generally known;see for example, Tzalis et al., Tetrahedron Lett. 36(34):6017-6020(1995); Tzalis et al., Tetrahedron Lett. 36(2):3489-3490 (1995); andTzalis et al., Chem. Communications (in press) 1996, all of which arehereby expressly incorporated by reference. See also the figures and theexamples, which describes the synthesis of metallocenes (in this case,ferrocene) attached via acetylene linkages to the bases.

In one embodiment, the nucleosides are made with transition metalligands, incorporated into a nucleic acid, and then the transition metalion and any remaining necessary ligands are added as is known in theart. In an alternative embodiment, the transition metal ion andadditional ligands are added prior to incorporation into the nucleicacid.

Once the nucleic acids of the invention are made, with a covalentlyattached attachment linker (i.e. either an insulator or a conductiveoligomer), the attachment linker is attached to the electrode. Themethod will vary depending on the type of electrode used. As isdescribed herein, the attachment linkers are generally made with aterminal “A” linker to facilitate attachment to the electrode. For thepurposes of this application, a sulfur-gold attachment is considered acovalent attachment.

In a preferred embodiment, conductive oligomers, insulators, andattachment linkers are covalently attached via sulfur linkages to theelectrode. However, surprisingly, traditional protecting groups for useof attaching molecules to gold electrodes are generally not ideal foruse in both synthesis of the compositions described herein and inclusionin oligonucleotide synthetic reactions. Accordingly, the presentinvention provides novel methods for the attachment of conductiveoligomers to gold electrodes, utilizing unusual protecting groups,including ethylpyridine, and trimethylsilylethyl as is depicted in theFigures. However, as will be appreciated by those in the art, when theconductive oligomers do not contain nucleic acids, traditionalprotecting groups such as acetyl groups and others may be used. SeeGreene et al., supra.

This may be done in several ways. In a preferred embodiment, the subunitof the conductive oligomer which contains the sulfur atom for attachmentto the electrode is protected with an ethyl-pyridine ortrimethylsilylethyl group. For the former, this is generally done bycontacting the subunit containing the sulfur atom (preferably in theform of a sulfhydryl) with a vinyl pyridine group or vinyltrimethylsilylethyl group under conditions whereby an ethylpyridinegroup or trimethylsilylethyl group is added to the sulfur atom.

This subunit also generally contains a functional moiety for attachmentof additional subunits, and thus additional subunits are attached toform the conductive oligomer. The conductive oligomer is then attachedto a nucleoside, and additional nucleosides attached. The protectinggroup is then removed and the sulfur-gold covalent attachment is made.Alternatively, all or part of the conductive oligomer is made, and theneither a subunit containing a protected sulfur atom is added, or asulfur atom is added and then protected. The conductive oligomer is thenattached to a nucleoside, and additional nucleosides attached.Alternatively, the conductive oligomer attached to a nucleic acid ismade, and then either a subunit containing a protected sulfur atom isadded, or a sulfur atom is added and then protected. Alternatively, theethyl pyridine protecting group may be used as above, but removed afterone or more steps and replaced with a standard protecting group like adisulfide. Thus, the ethyl pyridine or trimethylsilylethyl group mayserve as the protecting group for some of the synthetic reactions, andthen removed and replaced with a traditional protecting group.

By “subunit” of a conductive polymer herein is meant at least the moietyof the conductive oligomer to which the sulfur atom is attached,although additional atoms may be present, including either functionalgroups which allow the addition of additional components of theconductive oligomer, or additional components of the conductiveoligomer. Thus, for example, when Structure 1 oligomers are used, asubunit comprises at least the first Y group.

A preferred method comprises 1) adding an ethyl pyridine ortrimethylsilylethyl protecting group to a sulfur atom attached to afirst subunit of a conductive oligomer, generally done by adding a vinylpyridine or trimethylsilylethyl group to a sulfhydryl; 2) addingadditional subunits to form the conductive oligomer; 3) adding at leasta first nucleoside to the conductive oligomer; 4) adding additionalnucleosides to the first nucleoside to form a nucleic acid; 5) attachingthe conductive oligomer to the gold electrode. This may also be done inthe absence of nucleosides.

The above method may also be used to attach insulator molecules to agold electrode.

In a preferred embodiment, a monolayer comprising conductive oligomers(and optionally insulators) i added to the electrode. Generally, thechemistry of addition is similar to or the same as the addition oconductive oligomers to the electrode, i.e. using a sulfur atom forattachment to a gold electrode, etc. Compositions comprising monolayersin addition to the conductive oligomers covalently attached to nucleicacids may be made in at least one of five ways: (1) addition of themonolayer, followed by subsequent addition of the attachmentlinker-nucleic acid complex; (2) addition of the attachmentlinker-nucleic acid complex followed by addition of the monolayer; (3)simultaneous addition of the monolayer and attachment linker-nucleicacid complex; (4) formation of a monolayer (using any of 1, or 3) whichincludes attachment linkers which terminate in a functional moietysuitable for attachment of a completed nucleic acid; or (5) formation ofa monolayer which includes attachment linkers which terminate in afunctional moiety suitable for nucleic acid synthesis, i.e. the nucleicacid is synthesized on the surface of the monolayer as is known in theart. Such suitable functional moieties include, but are not limited to,nucleosides, amino groups, carboxyl groups, protected sulfur moieties,or hydroxyl groups for phosphoramidite additions. The examples describethe formation of a monolayer on a golc electrode using the preferredmethod (1).

In a preferred embodiment, the nucleic acid is a peptide nucleic acid oranalog. In this embodiment, the invention provides peptide nucleic acidswith at least one covalently attached ETM or attachment linker. In apreferred embodiment, these moieties are covalently attached to anmonomeric subunit of the PNA. By “monomeric subunit of PNA” herein ismeant the —NH—CH₂CH₂—N(COCH₂-Base)-CH₂—CO monomer, or derivatives(herein included within the definition of “nucleoside”) of PNA. Forexample, the number of carbon atoms in the PNA backbone may be altered;see generally Nielsen et al., Cherr. Soc. Rev, 1997 page 73, whichdiscloses a number of PNA derivatives, herein expressly incorporated byreference. Similarly, the amide bond linking the base to the backbonemay be altered; phosphoramide and sulfuramide bonds may be used.Alternatively, the moieties are attached to an internal monomericsubunit. By “internal” herein is meant that the monomeric subunit is noteither the N-terminal monomeric subunit or the C-terminal monomericsubunit. In this embodiment, the moietie can be attached either to abase or to the backbone of the monomeric subunit. Attachment to the baseis done as outlined herein or known in the literature. In general, themoieties are added to a base which is then incorporated into a PNA asoutlined herein. The base may be either protected, as required forincorporation into the PNA synthetic reaction, or derivatized, to allowincorporation, either prior to the addition of the chemical substituentor afterwards. Protection and derivatization of the bases is shown inPCT US97/20014. The bases can then be incorporated into monomericsubunits.

In a preferred embodiment, the moieties are covalently attached to thebackbone of the PNA monomer. The attachment is generally to one of theunsubstituted carbon atoms of the monomeric subunit, preferably thea-carbon of the backbone, although attachment at either of the carbon 1or 2 positions, or the a-carbon of the amide bond linking the base tothe backbone may be done. In the case of PNA analogs, other carbons oratoms may be substituted as well. In a preferred embodiment moieties areadded at the a-carbon atoms, either to a terminal monomeric subunit oran internal one.

In this embodiment, a modified monomeric subunit is synthesized with anETM or an attachment linker, or a functional group for its attachment,and then the base is added and the modified monomer can be incorporatedinto a growing PNA chain.

Once generated, the monomeric subunits with covalently attached moietiesare incorporated into a PNA using the techniques outlined in Will etal., Tetrahedron 51(44)12069-12082 (1995), and Vanderlaan et al., Tett.Let. 38:2249-2252 (1997), both of which are hereby expresslyincorporated in their entirety. These procedures allow the addition ofchemical substituents to peptide nucleic acids without destroying thechemical substituents.

As will be appreciated by those in the art, electrodes may be made thathave any combination of nucleic acids, conductive oligomers andinsulators.

The compositions of the invention may additionally contain one or morelabels at any position. By “label” herein is meant an element (e.g. anisotope) or chemical compound that is attached to enable the detectionof the compound. Preferred labels are radioactive isotopic labels, andcolored or fluorescent dyes. The labels may be incorporated into thecompound at any position. In addition, the compositions of the inventionmay also contain other moieties such as cross-linking agents tofacilitat cross-linking of the target-probe complex. See for example,Lukhtanov et al., Nucl. Acids. Res. 24(4):683 (1996) and Tabone et al.,Biochem. 33:375 (1994), both of which are expressly incorporate byreference.

Once made, the compositions find use in a number of applications, asdescribed herein. In particular the compositions of the invention finduse in binding assays for the detection of target analytes, inparticular nucleic acid target sequences. As will be appreciated bythose in the art, electrodes can be made that have a single species ofbinding ligand, or multiple binding ligand species, i.e. in an arrayformat.

In addition, as outlined herein, the use of a solid support such as anelectrode enables the use of these assays in an array form. For example,the use of oligonucleotide arrays are well known in the art. Inaddition, techniques are known for “addressing” locations within anelectrode and for the surface modification of electrodes. Thus, in apreferred embodiment, arrays of different binding ligands, includingnucleic acids, are laid down on the electrode, each of which arecovalently attached to the electrode via an attachment linker. In thisembodiment, the number of different binding ligands may vary widely,from one to thousands, with from about 4 to about 100,000 beingpreferred, and from about 10 to about 10,000 being particularlypreferred.

Once the assay complexes of the invention are made, that minimallycomprise a target analyte and a label probe, detection proceeds withelectronic initiation. Without being limited by the mechanism or theory,detection is based on the transfer of electrons from the ETM to theelectrode, including via the “π-way”.

Detection of electron transfer, i.e. the presence of the ETMs, isgenerally initiated electronically, with voltage being preferred. Apotential is applied to the assay complex. Precise control andvariations in the applied potential can be via a potentiostat and eithera three electrode system (one reference, one sample (or working) and onecounter electrode) or a two electrode system (one sample and one counterelectrode). This allows matching of applied potential to peak potentialof the system which depends in part on the choice of ETMs and in part onthe conductive oligomer used, the composition and integrity of themonolayer, and what type of reference electrode is used. As describedherein, ferrocene is a preferred ETM.

In a preferred embodiment, a co-reductant or co-oxidant (collectively,co-redoxant) is used, as an additional electron source or sink. Seegenerally Sato et al., Bull. Chem., Soc. Jpn 66:1032 (1993); Uosaki etal., Electrochimica Acta 36:1799 (1991); and Alleman et al., J. Phys.Chem. 100:17050 (1996); all of which are incorporated by reference. Insome cases, a copper electrode is used, which serves as a catalyticelectrode, creating co-reductants or co-oxidants in situ. See Fungal etal. Anal. Chem. 69:4828 (1997), incorporated herein by reference.

In a preferred embodiment, an input electron source in solution is usedin the initiation of electron transfer, preferably when initiation anddetection are being done using DC current or at AC frequencies wherediffusion is not limiting. In general, as will be appreciated by thosein the art, preferred embodiments utilize monolayers that contain aminimum of “holes”, such that short-circuiting of the system is avoided.This may be done in several general ways. In a preferred embodiment, aninput electron source is used that has a lower or similar redoxpotential than the ETM of the label probe. Thus, at voltages above theredox potential of the input electron source, both the ETM and the inputelectron source are oxidized and can thus donate electrons; the ETMdonates an electron to the electrode and the input source donates to theETM. For example, ferrocene, as a ETM attached to the compositions ofthe invention as described in the examples, has a redox potential ofroughly 200 mV in aqueous solution (which can change significantlydepending on what the ferrocene is bound to, the manner of the linkageand the presence of any substitution groups). Ferrocyanide, an electronsource, has a redox potential of roughly 200 mV as well (in aqueoussolution). Accordingly, at or above voltages of roughly 200 mV,ferrocene is converted to ferricenium, which then transfers an electronto the electrode. Now the ferricyanide can be oxidized to transfer anelectron to the ETM. In this way, the electron source (or co-reductant)serves to amplify the signal generated in the system, as the electronsource molecules rapidly and repeatedly donate electrons to the ETMattached to the nucleic acid. The rate of electron donation oracceptance will be limited by the rate of diffusion of the co-reductant,the electron transfer between the co-reductant and the ETM, which inturn is affected by the concentration and size, etc.

Alternatively, input electron sources that have lower redox potentialsthan the ETM are used. At voltages less than the redox potential of theETM, but higher than the redox potential of the electron source, theinput source such as ferrocyanide is unable to be oxided and thus isunable to donate an electron to the ETM; i.e. no electron transferoccurs. Once ferrocene is oxidized, then there is a pathway for electrontransfer.

In an alternate preferred embodiment, an input electron source is usedthat has a higher redox potential than the ETM of the label probe. Forexample, luminol, an electron source, has a redox potential of roughly720 mV. At voltages higher than the redox potential of the ETM, butlower than the redox potential of the electron source, i.e. 200-720 mV,the ferrocene is oxided, and transfers a single electron to theelectrode via the conductive oligomer. However, the ETM is unable toaccept any electrons from the luminol electron source, since thevoltages are less than the redox potential of the luminol. However, ator above the redox potential of luminol, the luminol then transfers anelectron to the ETM, allowing rapid and repeated electron transfer. Inthis way, the electron source (or co-reductant) serves to amplify thesignal generated in the system, as the electron source molecules rapidlyand repeatedly donate electrons to the ETM of the label probe.

Luminol has the added benefit of becoming a chemiluminiscent speciesupon oxidation (see Jirka et al., Analytica Chimica Acta 284:345(1993)), thus allowing photo-detection of electron transfer from the ETMto the electrode. Thus, as long as the luminol is unable to contact theelectrode directly, i.e. in the presence of the SAM such that there isno efficient electron transfer pathway to the electrode, luminol canonly be oxidized by transferring an electron to the ETM on the labelprobe. When the ETM is not present, i.e. when the target sequence is nothybridized to the composition of the invention, luminol is notsignificantly oxidized, resulting in a low photon emission and thus alow (if any) signal from the luminol. In the presence of the target, amuch larger signal is generated. Thus, the measure of luminol oxidationby photon emission is an indirect measurement of the ability of the ETMto donate electrons to the electrode. Furthermore, since photondetection is generally more sensitive than electronic detection, thesensitivity of the system may be increased. Initial results suggest thatluminescence may depend on hydrogen peroxide concentration, pH, andluminol concentration, the latter of which appears to be non-linear.

Suitable electron source molecules are well known in the art, andinclude, but are not limited to, ferricyanide, and luminol.

Alternatively, output electron acceptors or sinks could be used, i.e.the above reactions could be run in reverse, with the ETM such as ametallocene receiving an electron from the electrode, converting it tothe metallicenium, with the output electron acceptor then accepting theelectron rapidly and repeatedly. In this embodiment, cobalticenium isthe preferred ETM.

The presence of the ETMs at the surface of the monolayer can be detectedin a variety of ways. A variety of detection methods may be used,including, but not limited to, optical detection (as a result ofspectra) changes upon changes in redox states), which includesfluorescence, phosphorescence, luminiscence, chemiluminescence,electrochemiluminescence, and refractive index; and electronicdetection, including, but not limited to, amperommetry, voltammetry,capacitance and impedence. These methods include time or frequencydependent methods based on AC or DC currents, pulsed methods, lock-intechniques, filtering (high pass, low pass, band pass), andtime-resolved techniques including time-resolved fluoroscence.

In one embodiment, the efficient transfer of electrons from the ETM tothe electrode results in stereotyped changes in the redox state of theETM. With many ETMs including the complexes of ruthenium containingbipyridine, pyridine and imidazole rings; these changes in redox stateare associated with changes in spectral properties. Significantdifferences in absorbance are observed between reduced and oxidizedstates for these molecules. See for example Fabbrizzi et al., Chem. Soc,Rev, 1995 pp 197-202). These differences can be monitored using aspectrophotometer or simple photomultiplier tube device.

In this embodiment, possible electron donors and acceptors include allthe derivatives listed above for photoactivation or initiation.Preferred electron donors and acceptors have characteristically largespectral changes upon oxidation and reduction resulting in, highlysensitive monitoring of electron transfer. Such examples includeRu(NH₃)₄py and Ru(bpy)₂im as preferred examples. It should be understoodthat only the donor or acceptor that is being monitored by absorbanceneed have ideal spectral characteristics.

In a preferred embodiment, the electron transfer is detectedfluorometrically. Numerous transition metal complexes, including thoseof ruthenium, have distinct fluorescence properties. Therefore, thechange in redox state of the electron donors and electron acceptorsattached to the nucleic acid can be monitored very sensitively usingfluorescence, for example with Ru(4,7-biphenyl₂-phenanthroline)₃ ²⁺. Theproduction of this compound can be easily measured using standardfluorescence assay techniques. For example, laser induced fluorescencecan be recorded in a standard single cell fluorimeter, a flow through“on-line” fluorimeter (such as those attached to a chromatographysystem) or a multi-sample “plate-reader” similar to those marketed for96-well immuno assays.

Alternatively, fluorescence can be measured using fiber optic sensorswith nucleic acid probes in solution or attached to the fiber optic.Fluorescence is monitored using a photomultiplier tube or other lightdetection instrument attached to the fiber optic. The advantage of thissystem is the extremely small volumes of sample that can be assayed.

In addition, scanning fluorescence detectors such as the FluorImagersold by Molecular Dynamics are ideally suited to monitoring thefluorescence of modified nucleic acid molecules arrayed on solidsurfaces. The advantage of this system is the large number of electrontransfer probes that can be scanned at once using chips covered withthousands of distinct nucleic acid probes.

Many transition metal complexes display fluorescence with large Stokesshifts. Suitable examples include bis- and trisphenanthroline complexesand bis- and trisbipyridyl complexes of transition metals such asruthenium (see Juris, A., Balzani, V., et. al. Coord. Chem. Rev., V. 84,p. 85-277, 1988). Preferred examples display efficient fluorescence(reasonably high quantum yields) as well as low reorganization energies.These include Ru(4,7-biphenyl₂-phenanthroline)₃ ²⁺,Ru(4,4′-diphenyl-2,2′ bipyridine)₃ ²⁺ and platinum complexes (seeCummings et al., J. Am. Chem. Soc. 118:1949-1960 (1996), incorporated byreference). Alternatively, a reduction in fluorescence associated withhybridization can be measured using these systems.

In a further embodiment, electrochemiluminescence is used as the basisof the electron transfer detection. With some ETMs such asRu²⁺(bpy)_(3r) direct luminescence accompanies excited state decay.Changes in this property are associated with nucleic acid hybridizationand can be monitored with a simple photomultiplier tube arrangement (seeBlackburn, G. F. Olin, Chem. 37: 1534-1539 (1991); and Juris et al.,supra.

In a preferred embodiment, electronic detection is used, includingamperommetry, voltammetry, capacitance, and impedence. Suitabletechniques include, but are not limited to, electrogravimetry;coulometry (including controlled potential coulometry and constantcurrent coulometry); voltametry (cyclic voltametry, pulse voltametry,(normal pulse voltametry, square wave voltametry, differential pulsevoltametry, Osteryoung square wave voltametry, and coulostatic pulsetechniques); stripping analysis (aniodic stripping analysis, cathiodicstripping analysis, square wave stripping voltammetry); conductancemeasurements (electrolytic conductance, direct analysis); time-dependentelectrochemical analyses (chronoamperometry, chronopotentiometry, cyclicchronopotentiometry and amperometry, AC polography, chronogalvametry,and chronocoulometry); AC impedance measurement; capacitancemeasurement; AC voltametry; and photoelectrochemistry.

In a preferred embodiment, monitoring electron transfer is viaamperometric detection. This method of detection involves applying apotential (as compared to a separate reference electrode) between thenucleic acid-conjugated electrode and a reference (counter) electrode inthe sample containing target genes of interest. Electron transfer ofdiffering efficiencies is induced in samples in the presence or absenceof target nucleic acid; that is, the presence or absence of the targetnucleic acid, and thus the label probe, can result in differentcurrents.

The device for measuring electron transfer amperometrically involvessensitive current detection and includes a means of controlling thevoltage potential, usually a potentiostat. This voltage is optimizedwith reference to the potential of the electron donating complex on thelabel probe. Possible electron donating complexes include thosepreviously mentioned with complexes of iron, osmium, platinum, cobalt,rhenium and ruthenium being preferred and complexes of iron being mostpreferred.

In a preferred embodiment, alternative electron detection modes areutilized. For example, potentiometric (or voltammetric) measurementsinvolve non-faradaic (no net current flow) processes and are utilizedtraditionally in pH and other ion detectors. Similar sensors are used tomonitor electron transfer between the ETM and the electrode. Inaddition, other properties of insulators (such as resistance) and ofconductors (such as conductivity, impedance and capicitance) could beused to monitor electron transfer between ETM and the electrode.Finally, any system that generates a current (such as electron transfer)also generates a small magnetic field, which may be monitored in someembodiments.

It should be understood that one benefit of the fast rates of electrontransfer observed in the compositions of the invention is that timeresolution can greatly enhance the signal-to-noise results of monitorsbased on absorbance, fluorescence and electronic current. The fast ratesof electron transfer of the present invention result both in highsignals and stereotyped delays between electron transfer initiation andcompletion. By amplifying signals of particular delays, such as throughthe use of pulsed initiation of electron transfer and “lock-in”amplifiers of detection, and Fourier transforms.

In a preferred embodiment, electron transfer is initiated usingalternating current (AC) methods. Without being bound by theory, itappears that ETMs, bound to an electrode, generally respond similarly toan AC voltage across a circuit containing resistors and capacitors.Basically, any methods which enable the determination of the nature ofthese complexes, which act as a resistor and capacitor, can be used asthe basis of detection. Surprisingly, traditional electrochemicaltheory, such as exemplified in Laviron et al., J. Electroanal. Chem.97:135 (1979) and Laviron et al., J. Electroanal. Chem. 105:35 (1979),both of which are incorporated by reference, do not accurately model thesystems described herein, except for very small E_(AC) (less than 10 mV)and relatively large numbers of molecules. That is, the AC current (I)is not accurately described by Laviron's equation. This may be due inpart to the fact that this theory assumes an unlimited source and sinkof electrons, which is not true in the present systems.

Accordingly, alternate equations were developed, using the Nernstequation and first principles to develop a model which more closelysimulates the results. This was derived as follows. The Nernst equation,Equation 1 below, describes the ratio of oxidized (O) to reduced (R)molecules (number of molecules=n) at any given voltage and temperature,since not every molecule gets oxidized at the same oxidation potential.

$\begin{matrix}{{Equation}\mspace{14mu} 1} & \; \\{E_{D\; C} = {E_{0} + {\frac{RT}{nF}\ell\; n\frac{\lbrack O\rbrack}{\lbrack R\rbrack}}}} & (1)\end{matrix}$

E_(DC) is the electrode potential, E₀ is the formal potential of themetal complex, R is the gas constant, T is the temperature in degreesKelvin, n is the number of electrons transferred, F is faraday'sconstant, [0] is the concentration of oxidized molecules and [R] is theconcentration of reduced molecules.

The Nernst equation can be rearranged as shown in Equations 2 and 3: inr

$\begin{matrix}{{Equation}\mspace{14mu} 2} & \; \\{{E_{D\; C} - E_{0}} = {\frac{RT}{nF}\ell\; n\frac{\lbrack O\rbrack}{\lbrack R\rbrack}}} & (2)\end{matrix}$

E_(DC) is the DC component of the potential.

$\begin{matrix}{{Equation}\mspace{14mu} 3} & \; \\{{\exp\frac{nF}{RT}\left( {E_{D\; C} - E_{0}} \right)} = \frac{\lbrack O\rbrack}{\lbrack R\rbrack}} & (3)\end{matrix}$

Equation 3 can be rearranged as follows, using normalization of theconcentration to equal I for simplicity, as shown in Equations 4, 5 and6. This requires the subsequent multiplication by the total number ofmolecules.[O]+[R]=1  Equation 4[O]=1−[R]  Equation 5[R]=1−[0]  Equation 6

Plugging Equation 5 and 6 into Equation 3, and the fact that nF/RTequals 38.9 V⁻¹, for n=1, gives Equations 7 and 8, which define [0] and[R], respectively:

$\begin{matrix}{{Equation}\mspace{14mu} 7} & \; \\{\lbrack O\rbrack = \frac{\exp^{38.9{({E - E_{0}})}}}{1 + \exp^{38.9{({E - E_{0}})}}}} & (4) \\{{Equation}\mspace{14mu} 8} & \; \\{\lbrack R\rbrack = \frac{1}{1 + \exp^{38.9{({E - E_{0}})}}}} & (5)\end{matrix}$

Taking into consideration the generation of an AC faradaic current, theratio of [0]|[R] at any given potential must be evaluated. At aparticular E_(DC) with an applied E_(AC), as is generally describedherein, at the apex of the E_(AC) more molecules will be in the oxidizedstate, since the voltage on the surface is now (E_(DC)),+E_(AC)); at thebottom, more will be reduced since the voltage is lower. Therefore, theAC current at a given E_(DC) will be dictated by both the AC and DCvoltages, as well as the shape of the Nernstian curve. Specifically, ifthe number of oxidized molecules at the bottom of the AC cycle issubtracted from the amount at the top of the AC cycle, the total changein a given AC cycle is obtained, as is generally described by Equation9. Dividing by 2 then gives the AC amplitude.

$\begin{matrix}{i_{A\; C} = \frac{\begin{matrix}{\left( {{electrons}\mspace{14mu}{{at}\left\lbrack {E_{D\; C} + E_{A\; C}} \right\rbrack}} \right) -} \\\left( {{electrons}\mspace{14mu}{{at}\left\lbrack {E_{D\; C} - E_{A\; C}} \right\rbrack}} \right)\end{matrix}}{2}} & {{Equation}\mspace{14mu} 9}\end{matrix}$

Equation 10 thus describes the AC current which should result:Equation 10i _(AC)=C_(O)Fω½([O]_(E) _(DC) _(+E) _(AC) −[O]_(E) _(DC) _(−E) _(AC))  (6)

As depicted in Equation 11, the total AC current will be the number ofredox molecules C), times faraday's constant (F), times the AC frequency(w), times 0.5 (to take into account the AC amplitude), times the ratiosderived above in Equation 7. The AC voltage is approximated by theaverage, E_(AC) ^(2/π).

$\begin{matrix}{{AC} = {{\frac{C_{O}F\;\omega}{2}\left( \frac{\exp^{38.9{\lbrack{E_{D\; C} + \frac{2E_{A\; C}}{\pi} - E_{0}}\rbrack}}}{1 + \exp^{38.9{\lbrack{E_{D\; C} + \frac{2E_{A\; C}}{\pi} - E_{0}}\rbrack}}} \right)} - \frac{\exp^{38.9{\lbrack{E_{D\; C} - \frac{2E_{A\; C}}{\pi} - E_{0}}\rbrack}}}{1 + \exp^{38.9{\lbrack{E_{D\; C} - \frac{2E_{A\; C}}{\pi} - E_{0}}\rbrack}}}}} & {{Equation}\mspace{14mu} 11}\end{matrix}$

Using Equation 11, simulations were generated using increasingoverpotential (AC voltage). FIG. 22A shows one of these simulations,while FIG. 22B depicts a simulation based on traditional theory. FIGS.23A and 23B depicts actual experimental data using the Fc-wire ofExample 7 plotted with the simulation, and shows that the model fits theexperimental data very well. In some cases the current is smaller thanpredicted, however this has been shown to be caused by ferrocenedegradation which may be remedied in a number of ways. However, Equation11 does not incorporate the effect of electron transfer rate nor ofinstrument factors. Electron transfer rate is important when the rate isclose to or lower than the applied frequency. Thus, the true i_(AC)should be a function of all three, as depicted in Equation 12.i _(AC) =f(Nernst factors)f(K _(ET))f(instrument factors)  Equation 12

These equations can be used to model and predict the expected ACcurrents in systems which use input signals comprising both AC and DCcomponents. As outlined above, traditional theory surprisingly does notmodel these systems at all, except for very low voltages.

In general, non-specifically bound label probes/ETMs show differences inimpedance (i.e. higher impedances) than when the label probes containingthe ETMs are specifically bound in the correct orientation. In apreferred embodiment, the non-specifically bound material is washedaway, resulting in an effective impedance of infinity. Thus, ACdetection gives several advantages as is generally discussed below,including an increase in sensitivity, and the ability to “filter out”background noise. In particular, changes in impedance (including, forexample, bulk impedance) as between non-specific binding ofETM-containing probes and target-specific assay complex formation may bemonitored.

Accordingly, when using AC initiation and detection methods, thefrequency response of the system changes as a result of the presence ofthe ETM. By “frequency response” herein is meant a modification ofsignals as a result of electron transfer between the electrode and theETM. This modification is different depending on signal frequency. Afrequency response includes AC currents at one or more frequencies,phase shifts, DC offset voltages, faradaic impedance, etc.

Once the assay complex including the target sequence and label probe ismade, a first input electrical signal is then applied to the system,preferably via at least the sample electrode (containing the complexesof the invention) and the counter electrode, to initiate electrontransfer between the electrode and the ETM. Three electrode systems mayalso be used, with the voltage applied to the reference and workingelectrodes. The first input signal comprises at least an AC component.The AC component may be of variable amplitude and frequency. Generally,for use in the present methods, the AC amplitude ranges from about 1 mVto about 1.1 V, with from about 10 mV to about 800 mV being preferred,and from about 10 mV to about 500 mV being especially preferred. The ACfrequency ranges from about 0.01 Hz to about 100 MHz, with from about 10Hz to about 10 MHz being preferred, and from about 100 Hz to about 20MHz being especially preferred.

The use of combinations of AC and DC signals gives a variety ofadvantages, including surprising sensitivity and signal maximization.

In a preferred embodiment, the first input signal comprises a DCcomponent and an AC component. That is, a DC offset voltage between thesample and counter electrodes is swept through the electrochemicalpotential of the ETM (for example, when ferrocene is used, the sweep isgenerally from 0 to 500 mV) (or alternatively, the working electrode isgrounded and the reference electrode is swept from 0 to −500 mV). Thesweep is used to identify the DC voltage at which the maximum responseof the system is seen. This is generally at or about the electrochemicalpotential of the ETM. Once this voltage is determined, either a sweep orone or more uniform DC offset voltages may be used. DC offset voltagesof from about −1 V to about +1.1 V are preferred, with from about −500mV to about +800 mV being especially preferred, and from about −300 mVto about 500 mV being particularly preferred. In a preferred embodiment,the DC offset voltage is not zero. On top of the DC offset voltage, anAC signal component of variable amplitude and frequency is applied. Ifthe ETM is present, and can respond to the AC perturbation, an ACcurrent will be produced due to electron transfer between the electrodeand the ETM.

For defined systems, it may be sufficient to apply a single input signalto differentiate between the presence and absence of the ETM (i.e. thepresence of the target sequence) nucleic acid. Alternatively, aplurality of input signals are applied. As outlined herein, this maytake a variety of forms, including using multiple frequencies, multipleDC offset voltages, or multiple AC amplitudes, or combinations of any orall of these.

Thus, in a preferred embodiment, multiple DC offset voltages are used,although as outlined above, DC voltage sweeps are preferred. This may bedone at a single frequency, or at two or more frequencies.

In a preferred embodiment, the AC amplitude is varied. Without beingbound by theory, it appears that increasing the amplitude increases thedriving force. Thus, higher amplitudes, which result in higheroverpotentials give faster rates of electron transfer. Thus, generally,the same system gives an improved response (i.e. higher output signals)at any single frequency through the use of higher overpotentials at thatfrequency. Thus, the amplitude may be increased at high frequencies toincrease the rate of electron transfer through the system, resulting ingreater sensitivity. In addition, this may be used, for example, toinduce responses in slower systems such as those that do not possessoptimal spacing configurations.

In a preferred embodiment, measurements of the system are taken at atleast two separate amplitudes or overpotentials, with measurements at aplurality of amplitudes being preferred. As noted above, changes inresponse as a result of changes in amplitude may form the basis ofidentification, calibration and quantification of the system. Inaddition, one or more AC frequencies can be used as well.

In a preferred embodiment, the AC frequency is varied. At differentfrequencies, different molecules respond in different ways. As will beappreciated by those in the art, increasing the frequency generallyincreases the output current. However, when the frequency is greaterthan the rate at which electrons may travel between the electrode andthe ETM, higher frequencies result in a loss or decrease of outputsignal. At some point, the frequency will be greater than the rate ofelectron transfer between the ETM and the electrode, and then the outputsignal will also drop.

In one embodiment, detection utilizes a single measurement of outputsignal at a single frequency. That is, the frequency response of thesystem in the absence of target sequence, and thus the absence of labelprobe containing ETMs, can be previously determined to be very low at aparticular high frequency. Using this information, any response at aparticular frequency, will show the presence of the assay complex. Thatis, any response at a particular frequency is characteristic of theassay complex. Thus, it may only be necessary to use a single input highfrequency, and any changes in frequency response is an indication thatthe ETM is present, and thus that the target sequence is present.

In addition, the use of AC techniques allows the significant reductionof background signals at any single frequency due to entities other thanthe ETMs, i.e. “locking out” or “filtering” unwanted signals. That is,the frequency response of a charge carrier or redox active molecule insolution will be limited by its diffusion coefficient and chargetransfer coefficient. Accordingly, at high frequencies, a charge carriermay not diffuse rapidly enough to transfer its charge to the electrode,and/or the charge transfer kinetics may not be fast enough. This isparticularly significant in embodiments that do not have goodmonolayers, i.e. have partial or insufficient monolayers, i.e. where thesolvent is accessible to the electrode. As outlined above, in DCtechniques, the presence of “holes” where the electrode is accessible tothe solvent can result in solvent charge carriers “short circuiting” thesystem, i.e. the reach the electrode and generate background signal.However, using the present AC techniques, one or more frequencies can bechosen that prevent a frequency response of one or more charge carriersin solution, whether or not a monolayer is present. This is particularlysignificant since many biological fluids such as blood containsignificant amounts of redox active molecules which can interfere withamperometric detection methods.

In a preferred embodiment, measurements of the system are taken at atleast two separate frequencies, with measurements at a plurality offrequencies being preferred. A plurality of frequencies includes a scan.For example, measuring the output signal, e.g., the AC current, at a lowinput frequency such as 1-20 Hz, and comparing the response to theoutput signal at high frequency such as 10-100 kHz will show a frequencyresponse difference between the presence and absence of the ETM. In apreferred embodiment, the frequency response is determined at at leasttwo, preferably at least about five, and more preferably at least aboutten frequencies.

After transmitting the input signal to initiate electron transfer, anoutput signal is received or detected. The presence and magnitude of theoutput signal will depend on a number of factors, including theoverpotential/amplitude of the input signal; the frequency of the inputAC signal; the composition of the intervening medium; the DC offset; theenvironment of the system; the nature of the ETM; the solvent; and thetype and concentration of salt. At a given input signal, the presenceand magnitude of the output signal will depend in general on thepresence or absence of the ETM, the placement and distance of the ETMfrom the surface of the monolayer and the character of the input signal.In some embodiments, it may be possible to distinguish betweennon-specific binding of label probes and the formation of targetspecific assay complexes containing label probes, on the basis ofimpedance.

In a preferred embodiment, the output signal comprises an AC current. Asoutlined above, the magnitude of the output current will depend on anumber of parameters. By varying these parameters, the system may beoptimized in a number of ways.

In general, AC currents generated in the present invention range fromabout 1 femptoamp to about 1 milliamp, with currents from about 50femptoamps to about 100 microamps being preferred, and from about 1picoamp to about 1 microamp being especially preferred.

In a preferred embodiment, the output signal is phase shifted in the ACcomponent relative to the input signal. Without being bound by theory,it appears that the systems of the present invention may be sufficientlyuniform to allow phase-shifting based detection. That is, the complexbiomolecules of the invention through which electron transfer occursreact to the AC input in a homogeneous manner, similar to standardelectronic components, such that a phase shift can be determined. Thismay serve as the basis of detection between the presence and absence ofthe ETM, and/or differences between the presence of target-specificassay complexes comprising label probes and non-specific binding of thelabel probes to the system components.

The output signal is characteristic of the presence of the ETM; that is,the output signal is characteristic of the presence of thetarget-specific assay complex comprising label probes and ETMs. In apreferred embodiment, the basis of the detection is a difference in thefaradaic impedance of the system as a result of the formation of theassay complex. Faradaic impedance is the impedance of the system betweenthe electrode and the ETM. Faradaic impedance is quite different fromthe bulk or dielectric impedance, which is the impedance of the bulksolution between the electrodes. Many factors may change the faradaicimpedance which may not effect the bulk impedance, and vice versa. Thus,the assay complexes comprising the nucleic acids in this system have acertain faradaic impedance, that will depend on the distance between theETM and the electrode, their electronic properties, and the compositionof the intervening medium, among other things. Of importance in themethods of the invention is that the faradaic impedance between the ETMand the electrode is signficantly different depending on whether thelabel probes containing the ETMs are specifically or non-specificallybound to the electrode.

Accordingly, the present invention further provides electronic devicesor apparatus for the detection of analytes using the compositions of theinvention. The apparatus includes a test chamber for receiving a samplesolution which has at least a first measuring or sample electrode, and asecond measuring or counter electrode. Three electrode systems are alsouseful. The first and second measuring electrodes are in contact with atest sample receiving region, such that in the presence of a liquid testsample, the two electrophoresis electrodes may be in electrical contact.

In a preferred embodiment, the apparatus also includes detectionelectrodes comprising a single stranded nucleic acid capture probecovalently attached via an attachment linker, and a monolayer comprisingconductive oligomers, such as are described herein.

The apparatus further comprises an AC voltage source electricallyconnected to the test chamber; that is, to the measuring electrodes.Preferably, the AC voltage source is capable of delivering DC offsetvoltage as well.

In a preferred embodiment, the apparatus further comprises a processorcapable of comparing the input signal and the output signal. Theprocessor is coupled to the electrodes and configured to receive anoutput signal, and thus detect the presence of the target nucleic acid.

Thus, the compositions of the present invention may be used in a varietyof research, clinical, quality control, or field testing settings.

In a preferred embodiment, the probes are used in genetic diagnosis. Forexample, probes can be made using the techniques disclosed herein todetect target sequences such as the gene for nonpolyposis colon cancer,the BRCA1 breast cancer gene, P53, which is a gene associated with avariety of cancers, the Apo E4 gene that indicates a greater risk ofAlzheimer's disease, allowing for easy presymptomatic screening ofpatients, mutations in the cystic fibrosis gene, or any of the otherswell known in the art.

In an additional embodiment, viral and bacterial detection is done usingthe complexes of the invention. In this embodiment, probes are designedto detect target sequences from a variety of bacteria and viruses. Forexample, current blood-screening techniques rely on the detection ofantiHIV antibodies. The methods disclosed herein allow for directscreening of clinical samples to detect HIV nucleic acid sequences,particularly highly conserved HIV sequences. In addition, this allowsdirect monitoring of circulating virus within a patient as an improvedmethod of assessing the efficacy of anti-viral therapies. Similarly,viruses associated with leukemia, HTLV-I and HTLV-II, may be detected inthis way. Bacterial infections such as tuberculosis, clymidia and othersexually transmitted diseases, may also be detected.

In a preferred embodiment, the nucleic acids of the invention find useas probes for toxic bacteria in the screening of water and food samples.For example, samples may be treated to lyse the bacteria to release itsnucleic acid, and then probes designed to recognize bacterial strains,including, but not limited to, such pathogenic strains as, Salmonella,Campylobacter, Vibrio cholerae, Leishmania, enterotoxic strains of E.coli, and Legionnaire's disease bacteria. Similarly, bioremediationstrategies may be evaluated using the compositions of the invention.

In a further embodiment, the probes are used for forensic “DNAfingerprinting” to match crime-scene DNA against samples taken fromvictims and suspects.

In an additional embodiment, the probes in an array are used forsequencing by hybridization.

Thus, the present invention provides for extremely specific andsensitive probes, which may, in some embodiments, detect targetsequences without removal of unhybridized probe. This will be useful inthe generation of automated gene probe assays.

Alternatively, the compositions of the invention are useful to detectsuccessful gene amplification in PCR, thus allowing successful PCRreactions to be an indication of the presence or absence of a targetsequence. PCR may be used in this manner in several ways. For example,in one embodiment, the PCR reaction is done as is known in the art, andthen added to a composition of the invention comprising the targetnucleic acid with a ETM, covalently attached to an electrode via aconductive oligomer with subsequent detection of the target sequence.Alternatively, PCR is done using nucleotides labelled with a ETM, eitherin the presence of, or with subsequent addition to, an electrode with aconductive oligomer and a target nucleic acid. Binding of the PCRproduct containing ETMs to the electrode composition will allowdetection via electron transfer. Finally, the nucleic acid attached tothe electrode via a conductive polymer may be one PCR primer, withaddition of a second primer labelled with an ETM. Elongation results indouble stranded nucleic acid with a ETM and electrode covalentlyattached. In this way, the present invention is used for PCR detectionof target sequences.

In a preferred embodiment, the arrays are used for mRNA detection. Apreferred embodiment utilizes either capture probes or capture extenderprobes that hybridize close to the 3′ polyadenylation tail of the mRNAs.This allows the use of one species of target binding probe fordetection, i.e. the probe contains a poly-T portion that will bind tothe poly-A tail of the mRNA target. Generally, the probe will contain asecond portion, preferably non-poly-T, that will bind to the detectionprobe (or other probe). This allows one target-binding probe to be made,and thus decreases the amount of different probe synthesis that is done.

In a preferred embodiment, the use of restriction enzymes and ligationmethods allows the creation of “universal” arrays. In this embodiment,monolayers comprising capture probes that comprise restrictionendonuclease ends, as is generally depicted in FIG. 6. By utilizingcomplementary portions of nucleic acid, while leaving “sticky ends”, anarray comprising any number of restriction endonuclease sites is made.Treating a target sample with one or more of these restrictionendonucleases allows the targets to bind to the array. This can be donewithout knowing the sequence of the target. The target sequences can beligated, as desired, using standard methods such as ligases, and thetarget sequence detected, using either standard labels or the methods ofthe invention.

The present invention provides methods which can result in sensitivedetection of nucleic acids. In a preferred embodiment, less than about10×10⁶ molecules are detected, with less than about 10×10⁶ beingpreferred, less than 10×10⁴ being particularly preferred, less thanabout 10×10³ being especially preferred, and less than about 10×10²being most preferred. As will be appreciated by those in the art, thisassumes a 1:1 correlation between target sequences and reportermolecules; if more than one reporter molecule (i.e. electron transfermoeity) is used for each target sequence, the sensitivity will go up.

While the limits of detection are currently being evaluated, based onthe published electron transfer rate through DNA, which is roughly 1×10⁶electrons/sec/duplex for an 8 base pair separation (see Meade et al.,Angw. Chem. Eng. Ed., 34:352 (1995)) and high driving forces, ACfrequencies of about 100 kHz should be possible. As the preliminaryresults show, electron transfer through these systems is quiteefficient, resulting in nearly 100×10³ electrons/sec, resulting inpotential femptoamp sensitivity for very few molecules.

As will be appreciated by those in the art, the modules of the inventioncan be configured in a variety of ways, depending on the number and sizeof samples, and the number and type of desired manipulations. Severalpreferred embodiments are shown in the Figures.

As outlined herein, the devices of the invention can be used incombination with apparatus for delivering and receiving fluids to andfrom the devices. The apparatus can include a “nesting site” forplacement of the device(s) to hold them in place and for registeringinlet and outlet ports, if present. The apparatus may also include pumps(“off chip pumps”), and means for viewing the contents of the devices,including microscopes, cameras, etc. The apparatus may includeelectrical contacts in the nesting region which mate with contactsintegrated into the structure of the chip, to power heating orelectrophoresis, for example. The apparatus may be provided withconventional circuitry sensors in communication with sensors in thedevice for thermal regulation, for example for PCR thermal regulation.The apparatus may also include a computer system comprising amicroprocessor for control of the various modules of the system as wellas for data analysis.

All references cited herein are incorporated by reference in theirentirety.

I claim:
 1. A microfluidic device for the detection of a target analytein a fluid sample, said device comprising a solid support membercomprising: a) a sample inlet port; b) a configuration of electrodes forelectrokinetic movement of said sample on said solid support member uponapplication of a voltage; and c) a detection chamber comprising aplurality of detection electrodes positioned in said detection chamber,each comprising a binding ligand and a self assembled monolayer, whereinsaid detection chamber is in fluid connection to said sample inlet port;wherein said electrokinetic movement is electroosmotic movement orelectrohydrodynamic movement.
 2. The microfluidic device according toclaim 1 wherein said electrokinetic movement is electroosmotic movement.3. The microfluidic device according to claim 2 wherein said bindingligand is a nucleic acid.
 4. The microfluidic device according to claim2 wherein said binding ligand is a protein.
 5. The microfluidic deviceaccording to claim 1 wherein said electrokinetic movement iselectrohydrodynamic movement.
 6. A method for the detection of a targetanalyte in a sample, said method comprising: a) introducing said sampleto a sample inlet port of a microfluidic device according to claim 1; b)applying a voltage to said configuration of electrodes to move saidsample to said detection chamber; c) forming an attachment complexbetween said target analyte and at least one of said binding ligands,wherein said attachment complex comprises an electron transfer moietylabel; and d) detecting the presence of said label as an indication ofthe presence of said target.
 7. A method according to claim 6 whereinsaid electron transfer moiety label is a metallocene.
 8. A methodaccording to claim 7 wherein said metallocene is a ferrocene.
 9. Amethod according to claim 6 wherein said attachment complex comprisessaid target analyte, said capture binding ligand, and a solution bindingligand comprising said electron transfer moiety label.
 10. Amicrofluidic device for the detection of a target analyte in a fluidsample, said device comprising a solid support member comprising: a) asample inlet port; b) a configuration of electrodes for anelectrokinetic movement of said sample on said solid support member uponapplication of a voltage; c) a detection chamber comprising a pluralityof detection electrodes positioned in said detection chamber, eachcomprising a binding ligand and a self-assembled monolayer, wherein saiddetection chamber is in fluid connection to said sample inlet port;wherein said binding ligand is a nucleic acid or a protein.
 11. Themicrofluidic device of claim 10, wherein said electrokinetic movement iselectroosmotic movement.
 12. The microfluidic device of claim 10,wherein said electrokinetic movement is electrohydrodynamic movement.13. The microfluidic device of claim 10, wherein said binding ligand isa nucleic acid.
 14. The microfluidic device of claim 10, wherein saidbinding ligand is a protein.
 15. A method for the detection of a targetanalyte in a sample, said method comprising: a) introducing said sampleto a sample inlet port of a microfluidic device according to claim 10;b) applying a voltage to said configuration of electrodes to move saidsample to said detection chamber; c) forming an attachment complexbetween said target analyte and at least one of said binding ligands,wherein said attachment complex comprises an electron transfer moietylabel; and d) detecting the presence of said label as an indication ofthe presence of said target.
 16. The method of claim 15, wherein saidelectron transfer moiety label is a metallocene.
 17. The method of claim16, wherein said metallocene is a ferrocene.
 18. The method of claim 15,wherein said attachment complex comprises said target analyte, saidcapture binding ligand, and a solution binding ligand comprising saidelectron transfer moiety label.